Enzyme-Catalyzed Regioselective Acetylation of Functionalized Glycosides

Article abstract of DOI:10.1002/ejoc.202000696

Novozym 435 (N435) is an immobilized lipase (Candida antarctica lipase B) capable of catalyzing transesterfications in organic media. This paper describes how this enzyme can be used for regioselective acetylation of unprotected carbohydrates providing protected monosaccharide building blocks in only one step. Unprotected thiogylcosides with both gluco, xylo, and manno stereochemistry were tolerated by the enzyme and afforded the acetylated products in high yields. The regioselective acetylation was easily scaled up to a 5.0 gram scale and in most cases, no purification was needed. Several other glycosides, including 2-azido-2-deoxy glucosides, galactosides, and gluconolactones, were also acetylated and the scope and limitations using N435 has been uncovered.

Full text of DOI:10.1002/ejoc.202000696

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Enzyme Catalyzed Regioselective Acetylation of Functionalized
Glycosides
Authors: Thomas Holmstrøm and Christian Marcus Pedersen
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000696
Link to VoR: https://doi.org/10.1002/ejoc.202000696
01/2020

10.1002/ejoc.202000696
European Journal of Organic Chemistry
COMMUNICATION
Enzyme Catalyzed Regioselective Acetylation of Functionalized
Glycosides
Thomas Holmstrøm[a] and Christian Marcus Pedersen*[a]
[a]
Department of Chemistry
University of Copenhagen
Universitetsparken 5, 2100 København Ø, Denmark
E-mail: cmp@chem.ku.dk
Supporting information for this article is given via a link at the end of the document.
Abstract: Novozym 435 (N435) is an immobilized lipase (Candida
antarctica lipase B) capable of catalyzing transesterfications in
organic media. This paper describes how this enzyme can be used
for regioselective acetylation of unprotected carbohydrates providing
protected monosaccharide building blocks in only one step.
Unprotected thiogylcosides with both gluco, xylo and manno
stereochemistry was tolerated by the enzyme and afforded the
acetylated products in high yields. The regioselective acetylation was
easily scaled up to a 5.0 gram scale and in most cases no purification
was needed. Several other glycosides, including 2-azido-2-deoxy
glucosides, galactosides and gluconolactones, were also acetylated
and the scope and limitations using N435 have been uncovered.
making it easy to handle and recycle.[18] It can simply be filtered
of in order to separate the reaction mixture from the reagent giving
access to almost pure compounds without the need for further
purification. During the characterization of the enzyme, it was
found that a Ser-His-Asp catalytic triad was present at its active
site.[19] The immobilized enzyme has many applications ranging
from biofuel production to chiral resolution.[20,21] It has previously
been shown that methyl β-D-glucopyranoside can undergo a
regioselective acetylation on the 3-OH and 6-OH in an almost
quantitative yield in the presence of N435 and vinyl acetate.[22]
The substrate scope however seemed quite narrow and we
therefore wanted to investigate if this transformation could be
used on other more complicated, but more relevant, substrates
with different anomeric substituents such as phenols or thiols
(Scheme 1). This would give direct access to valuable glycosyl
donors and acceptors in one or very few steps.
Oligosaccharides play an important role in cell recognition and
signal
transduction
during
biological
events.[1]
The
oligosaccharides are composed of carbohydrate building blocks
that are, due to the complex nature of carbohydrates, often quite
tedious to synthesize. A typical oligosaccharide synthesis often
involves many steps of protective group chemistry in order to
achieve the desired regioselectively protected monosaccharides.
These steps often include both full and partial protections and
deprotections of the carbohydrates. Therefore, many methods
have been developed to protect the different hydroxyl groups of
carbohydrates with high regioselectivity using conventional
organic chemistry.[2] Among these methods are the regioselective
and reductive opening of 4,6- and 2,3-benzylidene acetals in
order to create free hydroxyl groups[3], hydrogen bond activated
acetylations[4] and regioselective stannylene acetal openings[5].
Furthermore is it possible to carefully silylate the 3-OH and 6-OH
of β-D-glucopyranosides due to the difference in reactivity of the
hydroxyl groups.[6,7] Common for all of these methods is the use
of specialized solvents for each single step in the protection
procedures of the carbohydrates rendering this strategy less
attractive in regards to sustainability and scalability. Furthermore,
many methods use stoichiometric amounts of reagents. Therefore
much work has been done on enzymatic reactions providing
regioselectively protected carbohydrates using catalytic amounts
of enzymes.[8–14] These enzymatic reactions fulfill the conditions
for sustainability albeit often with limitations in the scope and
selectivity. The enzymatic protective group chemistry has found
its use also on a preparative scale, but mainly for mono-protection
of the more reactive and accessible primary alcohol. [8,15–17] Many
enzymes have been shown to be very useful for this purpose,
however one enzyme stands out when reviewing the literature.
Novozym 435 (N435) is a commercially available immobilized
lipase produced by Novozymes. N435 is a lipase B from Candida
antarctica immobilized on a poly(methyl methacrylate) resin
Previous work
OH
O
OAc
O
OAc
Novozym 435
HO
HO
HO
AcO
OMe
OMe
OH
Unprotected
carbohydrate
OH
3,6-Protected
carbohydrate
This work
= SPh
OAc
X
OAc
Novozym 435
SEt
O
O
HO
AcO
X
X
HO
OPMP
On-octyl
=O
OH / N3
OH
3,6-Protected
carbohydrate
Unprotected carbohydrate:
xylo, gluco, galacto, manno
Scheme 1. . Diacetylation of methyl β-D-glucopyranoside in the presence of
Novozym 435. Previous work and the scope elaborated in this communication.
Phenyl thio-β-D-glucopyranoside 19 is a common building block
for oligosaccharide synthesis and can be prepared from
commercially available and inexpensive starting materials on a
large scale. Having a thiofunctionality on the anomeric position
makes the building block ideal for glycosyl donor synthesis and
hence as a participant in glycosylations. A further advantage of
having a thiofunctionality is its stability under most conditions
needed for protective group manipulations. When required it can
selectively be activated under very mild conditions using oxidizing
reagents.
The unprotected thioglycoside 19 was first tested as a substrate
in the reaction using the conditions presented by S. Riva et al.[22]
This afforded the desired product 1 after a prolonged reaction time
1
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10.1002/ejoc.202000696
European Journal of Organic Chemistry
COMMUNICATION
compared to the methyl pyranoside, in a yield of 81%. Alongside
the 3,6-O-diacetylated product 1, a small amount of the 2,6-
regioisomer 2 was formed. The two isomers could however be
separated by flash column chromatography. This initial study was
followed by a screening of solvents in order to investigate if the
conditions could be improved for this substrate (Table 1). From
this study it became clear that performing the reaction in neat vinyl
acetate resulted in a decrease in reaction time and improved yield
(Table 1). However, this also resulted in a less selective reaction
as more of the 2,6-anomer was formed. Interestingly, by using
acetonitrile, a larger decrease in regioselectivity was observed,
generating almost a 1:1 mixture of the 3,6- and the 2,6-
regioisomer. Performing the similar reaction in the absence of the
enzyme gave no conversion thereby indicating that the low
selectivity was not due to non-enzymatic acetylation of the
substrate. By using THF and the safer methyl tert-butyl ether
(MTBE)) a high selectivity was achieved. The use of MTBE was
particularly interesting as it gave rise to a great decrease in
reaction time compared to THF. Finally by using the green solvent
2-MeTHF,[23] an impressive yield and selectivity was achieved in
a matter of only 45 hours showing that this transformation can
have a good sustainability profile.
OAc
O
a)
b)
HO
AcO
SPh
OH
BzCl, TMEDA
4Å MS
TMSCl
Et3N
1
OAc
OAc
O
O
BzO
AcO
TMSO
AcO
SPh
SPh
OBz
OTMS
3
4
(75%)
(97%)
Benzoyl protection
TMS protection
Scheme 2. Protective groups manipulations using building block 1.
With large quantities of the new building block in hand, the two
remaining hydroxyl groups on the 2 and 4 position could be
protected with different protective groups. A benzoyl protection
was first carried out using benzoyl chloride and TMEDA.[24] This
reaction proceeded in a matter of 1 hour and the fully protected
carbohydrate
3
could be isolated using flash column
chromatography and in a good yield (Scheme 2, a). Silylation of
the two hydroxyl groups using TMSCl and triethylamine afforded
glucosyl donor 4 (Scheme 2, b) in high yields. Installing ether
protective groups were however not successful. Benzyl or allyl
groups could not be installed under strong basic conditions due
to migration of the acetyl groups. Furthermore, it was not possible
to install benzyl or allyl groups under acidic conditions using
benzyl or allyl trichloroacetimidates. A reductive benzylation
procedure starting from the TMS protected precursor 4, as
presented by Cai and coworkers, did not give rise to the desired
2,4-O-dibenzylated product.[25,26]
Table 1. Solvent screening for the acetylation of 19 using vinyl acetate and
Novozym 435.
OAc
(10 equiv.)
Novozym 435
OH
O
OAc
O
OAc
O
HO
HO
HO
AcO
HO
+
SPh
SPh
SPh
HO
OH
OH
OAc
Solvent
45 °C
19
1
2
3,6-regioisomer
2,6-regioisomer
Solvent
Time[a]
Crude yield
3,6-isomer[b]
2,6-isomer[b]
A series of glucopyranosides with different anomeric substituents,
all with β-stereochemistry, were then investigated as substrates
in the acetylation reaction. As standard conditions, the
pyranosides were treated with 20% (w/w) N435 in a 1:1 mixture
of vinyl acetate and dry THF (4 mL/100 mg) at 45°C. It was found
that the enzyme could tolerate both aliphatic (Table 2, Entry 2, 4,
5) and aromatic (Table 2, Entry 1, 3) substituents. In general, the
thioglucosides demanded (Entry 1-2) a longer reaction time
compared to the O-glucosides (Entry 3-5). Among the O-
glycosides the aliphatic substituents showed a greater reactivity
compared to the aromatic p-methoxyphenyl (PMP) substituent
(Entry 3). The 3,6-O-diacetylated glucopyranosides 9 and 10
were isolated in a quantitative yield after filtration of the reaction
mixture and evaporation of the solvent and the unreacted vinyl
acetate. The other entries required purification by column
chromatography. In all cases, the reaction could be stopped in a
matter of hours to give exclusively the 6-O-acetylated product.
These intermediates were however not isolated in this study.
Upon the first acetylation of the 6-OH, an acetylation of the 3-OH
would take place giving rise to the 3,6-O-diacetylated products.
This could indicate that the 3,6-regioselectivity is due to the
nucleophilicity of the different hydroxyl groups. It is known from
literature that β-glucopyranosides are most reactive on the 6-OH
position, then followed by the 3-OH.[6,7]
Neat
27h
71h
91h
91h
91h
40h
45h
Quant.
96%
86%
69%
89%
52%
0%[c]
88%
93%
14%
27%
11%
48%
0%[c]
12%
7%
Toluene
THF
Quant.
Quant.
0%[c]
Acetonitrile
Acetonitrile
MTBE
Quant.
Quant.
2-MeTHF
[a] Reactions were performed by dissolving compound 19 and vinyl acetate
in the indicated solvent (0.5 mL) together with N435 (20% w/w) at 45 °C. [b]
Determined by 1H NMR. [c] Performed without N435.
With these preliminary results in hand, an attempt to scale up the
reaction was performed. The conditions using THF as the solvent
was chosen as these gave rise to a higher selectivity and thereby
an easier purification. Upon scaling up, it was found that the use
of a 1:1 mixture of vinyl acetate and THF and a 460 mM
concentration of substrate gave the best results. It was
unproblematic to perform the reaction on a 5 gram scale
demonstrating the scalability of this approach. Purification was
done by filtering the reaction mixture though a plug of silica eluting
with a gradient of ethyl acetate and heptane. This afforded an
almost pure solution of the product and the minor isomer
alongside with the immobilized that could be reused.
2
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10.1002/ejoc.202000696
European Journal of Organic Chemistry
COMMUNICATION
Table 2. Acetylation of different β-D-glucopyranosides.
galactopyranoside 29 gave exclusively the 2,6-O-diacetylated
product 17 (Table 3, Entry 7). The acetylation of D-glucono-1,5-
lactone 30 proceeded in a lower yield of 48%, but with high
regioselectivity as only the 2,6-diacetylated compound could be
isolated (Table 3, Entry 8).
Entry
Substrate
OH
Time
Product
Yield[a]
1
OAc
O
78%[b]
140h
O
HO
HO
HO
SPh
SEt
SPh
SEt
AcO
OH
OH
19
1
Table 3. Acetylation of different carbohydrates.
2
3
OH
O
160h
OAc
O
67%
Entry
Substrate
Time
Product
Yield[a]
HO
AcO
HO
HO
1
OH
OH
O
HO
HO
HO
140h
AcO
HO
AcO
Quant.
OH
OH
O
7
6
SPh
SPh
23
11
OH
O
72h
24h
46h
OAc
O
73%
HO
HO
OH
O
OAc
O
HO
2
3
3h
93%
90%
OPMP
OMe
OPMP
OMe
AcO
HO
HO
HO
OH
OH
20
HO
8
N
N
3SPh
3SPh
12
24
4
5
OH
O
OAc
O
Quant.
Quant.
HO
HO
HO
OH
O
OAc
O
48h
AcO
HO
HO
HO
HO
OH
OH
SPh
SPh
SPh
SPh
21
9
N3
N3
13
25
OH
OAc
O
O
4
5
O
140h
71h
O
71%
HO
HO
HO
AcO
HO
HO
AcO
AcO
O
7
O
7
OH
OH
OH
OH
22
10
14
26
[a] Reactions were performed by using the standard acetylation procedure
presented in the SI. [b] Performed on a 5.0 gram scale.
O
O
Quant.
HO
HO
HO
HO
HO
27
AcO
15
OMe
OMe
Impressed by the broad scope of the enzymatic acetylation we
turned our attention to glycosyl donors with different
stereochemistry and two 2-azido-2-deoxy glucosyl donors.
Mannosyl donors with a 3,6-protective group pattern is of great
interest since 3,6-branched mannosides are a core structure of
many oligosaccharides.[27] Starting from the unprotected
mannosyl donor 23 it was possible to obtain a 3,6-O-diacetylation
in a quantitative yield to give 11 using the standard conditions. It
was also possible to selectively acetylate the 6-OH on both α- and
β-D-glucosyl donors with 2-azido-2-deoxy functionalities (Table 3,
Entry 2 and 3). In this example, the anomeric configuration did not
affect the regioselectivity of the reaction as only one acetylation
took place. Even with the prolonged reaction time presented in
Table 3, Entry 3, a second acetylation was not observed. Having
xylo stereochemistry led to either a selective 3,4-O-diacetylation
or a 2-O-acetylation depending on the anomeric configuration,
when subjecting xylopyranoside 26 or 27 to the acetylation
conditions (Table 3, Entry 4 and 5). The observed 3,4-
regioselectivity for the acetylation of β-D-xylopyranoside has
previously been presented by Fernández-Mayoralas and
coworkers using vinyl acetate in acetonitrile in the presence of
Lipase PS.[28] Galacto stereochemistry gave rise to a less
6
OH
22h
OH
Quant.
1:1.4
OH
O
OAc
O
16a
OMe
OMe
OMe
HO
AcO
OH
OH
+
28
OH
OAc
O
16b
HO
OAc
OH
OH
7
8
51h
91h
Quant.
48%
OH
O
OAc
O
HO
HO
HO
AcO
OMe
OMe
29
17
OH
O
OAc
O
HO
HO
HO
HO
O
O
OH
OAc
18
30
[a] Reactions were performed by using the standard acetylation procedure
presented in the SI.
Overall, many of the substrates showed great selectivity by using
the acetylation procedure optimized for the thioglucoside 19
presented earlier. The reaction time did vary depending on the
substrate and some reactions demanded as much as 160 hours
to complete. However, we are confident that these reaction times
can be optimized for each of the substrates.
regioselective
reaction.
When
treating
methyl
β-D-
galactopyranoside 28 to the acetylation conditions, a mixture of
two co-eluting, diacetylated compounds was formed (Table 3,
Entry 6). By NMR spectroscopy it was established that the mixture
contained the 3,6- and the 2,6-O-diacetylated galactopyranosides
in a 1:1.4 ratio respectively. Having an axial hydroxyl group on the
4 position either decrease the nucleophilicity of the 3-OH or
increase the nucleophilicity of the 2-OH which is commonly lower
than 3-OH in a β-D-glucopyranoside.[29,30] The low regioselectivity
could however be circumvented by having the α-configuration,
then the 2-OH is more readily acetylated, i.e. acetylation of α-D-
In conclusion we have shown that N435 can be used for the
preparation of useful building blocks for the synthesis of 3,6- or
2,4-branched oligosaccharides. It was found that THF, MTBE and
2-MeTHF worked very well as solvents and that the reaction is
scalable. The 3,6-O-diacetylated thioglucoside was subjected to
two other kinds of protective groups in order to afford a fully
protected glucosyl donor with a 3,6-protective group pattern.
Furthermore, it was found that many other carbohydrates could
3
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10.1002/ejoc.202000696
European Journal of Organic Chemistry
COMMUNICATION
be acetylated with a high degree of regioselectivity giving access
to distinct protective group patterns in only one step.
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Acknowledgements
We would like to thank Novozymes A/S and Jesper Brask are
thanked for providing the N435 used in this work. TH thanks the
Villum foundation for financial support.
Keywords: Regioselective, acetylation, site-selective, enzymatic
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4
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10.1002/ejoc.202000696
European Journal of Organic Chemistry
COMMUNICATION
Enzyme Catalyzed Regioselective Acetylation of Functionalized Glycosides
Gluco- Manno-
Galacto- Xylo-
•3,6-Regioselectivty
•High yields
•Gram scale
OAc
Vinyl
acetate
stereochemistry
= SPh
 435
X
Novozym
OAc
O
SEt
OPMP
On-octyl
=O
X
O
HO
HO
AcO
X
OH / N3
Enzymatic acetylation
OH
A wide range of functionalized mono-saccharides have been regioselectively 3,6-acetylated using a commercially available enzyme.
The enzyme tolerates different anomeric groups and stereochemistry of the sugar, making the method highly attractive for building
block synthesis. The reaction works under neat conditions and also with green solvents and hence halogenated solvents, heavy
metal reagents and multiple reactions are avoided.
5
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