Development of regioselective deacylation of peracetylated β-d-monosaccharides using lipase from Pseudomonas stutzeri under sustainable conditions

Article abstract of DOI:10.1039/c4ra10401c

The lipase-catalyzed regioselective deacylation of peracetylated pyranosides has been evaluated in biosolvents. Among the biocatalysts tested, lipase from Pseudomonas stutzeri showed the highest activity, displaying regiospecific activity towards the anomeric position. This lipase was also employed in the regioselective alcoholysis of peracetylated sugars in green solvents, contributing to improve the sustainability of the process. Yields up to 97% of the desired product with different biosolvents were found. These reactions took place without noticeable activity and with total regioselectivity, representing a considerable improvement over the use of an aqueous buffer or conventional organic solvents. Furthermore, scaled up reactions are feasible without losing catalytic action. In order to understand the role of these biosolvents in the enzyme's synthetic behaviour, molecular modelling and docking studies were performed in the presence of some selected biosolvents to conclude that the presence of biosolvents in the reaction media modifies the access of the alcohols to the enzymatic active site allowing the presence of small alcohols and not i-propyl and t-butyl residues in the alcohol. This journal is

Full text of DOI:10.1039/c4ra10401c

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Development of regioselective deacylation of
peracetylated b-D-monosaccharides using lipase
from Pseudomonas stutzeri under sustainable
conditions†
Cite this: RSC Adv., 2014, 4, 55495
a
a
b
c
c
a
*
M. Sandoval, P. Hoyos, A. Cortes, T. Bavaro, M. Terreni and M. J. Hernaiz
´
´
The lipase-catalyzed regioselective deacylation of peracetylated pyranosides has been evaluated in
biosolvents. Among the biocatalysts tested, lipase from Pseudomonas stutzeri showed the highest
activity, displaying regiospecific activity towards the anomeric position. This lipase was also employed in
the regioselective alcoholysis of peracetylated sugars in green solvents, contributing to improve the
sustainability of the process. Yields up to 97% of the desired product with different biosolvents were
found. These reactions took place without noticeable activity and with total regioselectivity, representing
a considerable improvement over the use of an aqueous buffer or conventional organic solvents.
Furthermore, scaled up reactions are feasible without losing catalytic action. In order to understand the
role of these biosolvents in the enzyme's synthetic behaviour, molecular modelling and docking studies
were performed in the presence of some selected biosolvents to conclude that the presence of
biosolvents in the reaction media modifies the access of the alcohols to the enzymatic active site
allowing the presence of small alcohols and not i-propyl and t-butyl residues in the alcohol.
Received 13th September 2014
Accepted 17th October 2014
DOI: 10.1039/c4ra10401c
www.rsc.org/advances
preparation of sugar intermediates using only acetyl for
protection of hydroxyl groups.6 Selectively acetylated sugars
Introduction
containing only one free position can be in fact easily used as
key intermediates in the preparation of different glycoder-
ivatives of biological interest.7–9
In this respect, lipases have been shown to be very versatile
enzymes displaying high regio-, chemo- and enantioselectivity
under mild reaction conditions and exhibiting very high
stability in organic medium.10–13 These enzymes have been
widespread employed in stereoselective hydrolysis of esters and
trans-esterication reactions.11,14–16
In particular, the regioselective hydrolysis of peracetylated
saccharides has been reported using different lipases, such as
those from Candida rugosa (CRL), lipase B from Candida ant-
arctica (CAL-B), Pseudomonas uorescens (PFL), Thermomyces
lanuginose (TLL) and Rhizomucor miehei (RML), leading to
diverse results depending on the enzyme, the immobilization
procedure used for the enzyme and substrate employed.8,14,17,18
Thus, the search for a bio-catalyst with high activity, regio-
and stereoselectivity towards a large number of substrates is an
important challenge. For example, CRL is active towards many
monosaccharides, allowing selective hydrolysis only in C-6
position,6,8 while immobilized Lecitase® (phospholipase A1) is
moderately selective for the anomeric position, C-6 or both
depending on the conducted immobilization.19
Carbohydrates play an important role in many molecular
recognition processes such as embryogenesis, neuronal prolif-
eration and apoptosis phenomena.1–5 As the understanding of
these biological functions increases, the need to develop more
efficient synthetic procedures for oligosaccharide production in
large quantities has become a major subject. The main limita-
tion in the synthesis of oligosaccharides is represented by the
preparation of sugar building blocks bearing only one free
hydroxyl group in the desired position. Traditionally, the
synthesis of mono-deprotected carbohydrates has been devel-
oped through multistep processes, involving different protect-
ing groups, yielding mixtures of products and, in some cases,
low yields.6–9 In this context, the use of regioselective enzymes
for the hydrolysis of per-acetylated sugars has been recently
proposed as
a simple and efficient procedure for the
aDepartment of Pharmaceutical and Organic Chemistry, Faculty of Pharmacy,
Complutense University of Madrid, Campus de Moncloa, 30100 Madrid, Spain.
E-mail: mjhernai@ucm.es; Fax: +34 9139 41822
b
´
´
Unidad de Bioinformatica. Centro de Biologıa Molecular “Severo Ochoa” (CBMSO),
´
´
CSIC, Universidad Autonoma de Madrid (UAM), C/Nicolas Cabrera 1, 28049,
Madrid, Spain
cDepartment of Drug Sciences and Italian Biocatalysis Center, University of Pavia, via
Taramelli 12, 27100 Pavia, Italy
Two approaches are mainly used for the asymmetric deacy-
lation of peracetylated sugars: hydrolysis, in reaction media
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra10401c
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normally containing some organic solvents to ensure substrate
solubility, and alcoholysis reaction, where the solvent is also the
reagent.12,17,20
The use of more sustainable solvents is a challenge in order
to develop environmentally friendly processes. In this sense,
green solvents must assemble the basic principles of green
chemistry,21–23 such as: less hazardous chemical synthesis, use
of safe solvents, use of renewable feedstock and design to
ensure a suitable degradation in the environment. Accordingly,
solvents derived from biomass (bio-solvents),24 such, as glycerol
or dimethyl amide derivatives may be considered as green
solvents.25–27 Besides, uorinated solvents are also considered
“green solvents” due to their particular properties (temperature
dependence solubility in organic–water media, low toxicity, and
low volatility).28–30 In addition, the employ of a biocatalytic
strategy in combination with green solvents, actively contrib-
utes to greatly enhance the sustainability of a process allowing
the development of preparative methods with high substrate
concentration.30–32
In this work, we have evaluated the activity of different
lipases in the selective hydrolysis of peracetylated pyranosides
in order to obtain different monosaccharides with a free
hydroxyl group at the anomeric position, useful as intermedi-
ates to obtain, previous chemical activation and glycosylation,
oligosaccharides of biological relevance. For developing an
efficient preparative green bio-process, suitable to replace the
toxic reagents (such as hydrazine acetate) and solvents currently
used in chemical deprotection of the anomeric position33 the
search of a selective bio-catalysts has been combined with the
use of green solvents. Thus, various “green” solvents have been
studied to achieve high substrate concentration, selectivity and
yields. In particular, the Pseudomonas stutzeri (PSL) was effi-
ciently employed in hydrolysis and alcoholysis of several
substrates and considered as catalyst for developing preparative
bio-processes in green solvents. The good results obtained with
this enzyme, improved compared with other lipases currently
used in the hydrolysis of peracetylated pyranosides, have been
explained by molecular modelling and docking studies.
Scheme 1 Lipase-catalyzed regioselective hydrolysis of pyranoses.
Concerning toxicity issues, there are some experimental data
pointing to glycerol-derived solvents as being low hazardous, For
instance, toxicities of glycerol based solvents like S5 and the closely
related 1,3-diethoxy-2-propanol (LD50 > 3000 mg kgꢀ1, oral in
mice)34 and 1,2,3-triethoxypropane (LD50 ¼ 660 mg kgꢀ1, intra-
peritoneal in mice)34 are very low, in some case even lower than
that of butanol (LD50 ¼ 2680 mg kgꢀ1, oral in mice). Fluorinated
solvents will be probably more toxic than aliphatic ones, but
lacking in experimental data for them it would be reasonable to
take the toxicities of compounds produced in their probable
decomposition, like triuoroethanol (LD50 ¼ 366 mg kgꢀ1, oral in
mice)35 triuoroacetic acid (LDlo ¼ 150 mg kgꢀ1, intraperitoneal in
mice)35 or 2,2,2-triuoroethyl ethyl ether (LD50 ¼ 5100 mg kgꢀ1
,
intraperitoneal in rats)36 as referents of the toxicity of these kinds
of solvents.
Moreover using these glycerol derivatives allow to reduce
some other hazardous aspects. For instance, they have low
volatility, so the concentration in the air using them is lower
than using other conventional solvents. Albeit toxicities of these
solvents are not well known at this moment it is clear they are
not extremely toxic, meeting one of the key requirements for
green applications.
Results and discussion
Screening of solvents and lipase activity
Lipases QLC, PSL and CAL-B were evaluated in order to deter-
mine their potential activity in the regioselective deacylation of
peracetylated sugar (Scheme 1). 1,2,3,4,6-Penta-O-acetyl-b-D-
galactopyranose (1b, see Scheme 1) was used as standard
substrate with several wet-solvents (10% v/v water). The
complete listing of the solvents used in this work is given in
Fig. 1.
Some physico-chemical parameters of the solvents used in
this work are gathered in Table S2 (ESI†). As can be seen, the
structural diversity of the glycerol derivatives results in a wide
range of hydrophobicities. Some of the glycerol-derived solvents
used, namely those bearing uorinated chains, display simul-
taneously both high hydrophobicity and hydrogen-bond donor
ability, a rather unusual combination in conventional organic
solvents.
Fig. 1 Molecular structure of the solvents S1 to S7.
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Table 1 Deacylation at the anomeric position of 1,2,3,4,6-penta-O-
acetyl-b-D-galactopyranose (1b) catalyzed by several lipases under
different solvents containing 10% v/v of water
in ESI†). In particular CAL-B provided much poorer results
compared with the process previously reported and performed
using this immobilized enzyme.18
It is worth of mention the good result obtained with PSL in
the regioselective deacylation of the anomeric carbon in
substrate 1b, as not many hydrolases have been described to
present high selectivity towards the hydrolysis of this position.
Thermomyces lanuginose (TLL), Aspergillus niger (ANL) and C.
antarctica lipases have been previously reported to provide
hydrolysis in the anomeric position of 1b but the type of
immobilization used for the enzymes was crucial to achieve
good yields in acceptable reaction times.18 All those enzymes
were almost inactive in the hydrolysis of 1a, showing poor
selectivity towards primary acetoxy group (C6 position) when
used in the hydrolysis of peracetylated glucopyranosides.8,18
The C-1 regioselective deacylation of gluco- and galacto
pyranosides has also been achieved by using PFL as bio-
catalyst,14 that belongs to Pseudomonas species, although the
selectivity of the reaction and, consequently, the yields ach-
ieved, were strongly dependent on the substrate. Hydrolysis of
the anomeric position of mannopyranoses, was instead repor-
ted with moderate yields by using the immobilized phospholi-
pase “Lecitase”, but this enzyme preferentially hydrolyzed the
primary acetoxy group of gluco- and galactopyranoses.19
Furthermore, it should be considered that almost all the
processes previously reported for the hydrolysis of pyranoses,
were performed in aqueous homogenous reaction media
(mainly using 20% v/v of acetonitrile)6 and this reaction media
strongly limited the operational range of substrate and product
concentrations. In this context, the use of an enzyme active
towards a wide range of peracetylated pyranosides, highly
selective for the anomeric position and stable and in organic
green solvents, could be considered extremely useful for
developing preparative chemo-enzymatic process for the
synthesis of oligosaccharides. Based on these assumptions PSL
was selected for further studies.
Entry
Enzyme
Solvent
Time (h)
1a conv.a (%)
1
2
3
4
5
6
7
8
9
10
CAL-Bb
CAL-Bb
QLCc
QLCc
PSLd
S1
S2
S1
S2
S1
S2
S3
S4
24
24
24
24
8
24
24
24
24
24
3
3
1
1
92
76
62
2
15
25
PSLd
PSLd
PSLd
PSLd
THF
2-MeTHF
PSLd
a
Determined by HPLC: Asahipak column NH2P50-4E (4.6 mm ꢁ 250
mm); ELSD detector; mobile phase CH3CN–H2O, 80 : 20 (v/v), ow
rate 0.8 mL minꢀ1
.
14.9 IU mgꢀ1 protein. 3.91 IU mgꢀ1 protein.
b
c
d
36.2 IU mgꢀ1 protein.
Results from this experiment are summarized in Table 1.
According to Table 1, PSL was the best enzyme to catalyze the
deacylation of 1b in wet-S1 as reaction medium, obtaining 92%
yield in 8 h (entry 5). The product was puried and the spec-
troscopic elucidation by NMR showed a regiospecic deacyla-
tion towards the anomeric position of the peracetylated
substrate. Complete conversion of substrate 1b into product 1a
(as a mixture of anomers a and b) was achieved without
formation of other products as detected by HPLC analysis.
In fact, as reported in Fig. 2, maximum conversion (96%) was
achieved aer 24 h and the reaction proceeded with a complete
regioselectivity, because all the consumed substrate was con-
verted in product 1a. Aer complete hydrolysis of 1b, the
concentration of the product remained constant (no further
hydrolysis was observed). Good yields (76%) were also observed
when S2 was employed (entry 6). With PSL different other
solvents were tested, although worst results were obtained
(Table 1). CAL-B and QLC did not display good activity in S1 and
S2 (Table 1) as well as in any other solvents tested (see Table S1
Hydrolysis of other peracetylated substrates using P. stutzeri
lipase
Accordingly, hydrolytic assays on some different peracetylated
substrates were carried out with PSL (Scheme 1), in the presence
of the solvents S1 and S2. In order to study the potential
selectivity of this biocatalyst towards a or b anomers, per-
acetylated a-glycopyranoses were also included in the study.
Thus, the following peracetylated monosaccharides were
employed as substrates: 1,2,3,4,6-penta-O-acetyl-a-D-gal-
actopyranose (1a), 1,2,3,4,6-penta-O-acetyl-b-D-glucopyranose
(2b), 1,2,3,4,6-penta-O-acetyl-a-D-glucopyranose (2a), 1,2,3,4,6-
penta-O-acetyl-b-D-mannopyranose (3b), 1,2,3,4,6-penta-O-
acetyl-a-D-mannopyranose (3a), 2-acetamido-2-deoxy-1,3,4,6,-
tetra-O-acetyl-b-D-glucopyranose (4b), 2-acetamido-2-deoxy-
1,3,4,6,-tetra-O-acetyl-a-D-glucopyranose (4a) 2-acetamido-2-
deoxy-1,3,4,6,-tetra-O-acetyl-b-galactopyranose (5b) and 2-acet-
amido-2-deoxy-1,3,4,6,-tetra-O-acetyl-a-D-galactopyranose (5a).
Results are summarized in Table 2.
Fig. 2 Kinetic of the regioselective deacylation of 1b catalyzed by PSL
in S1.
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Table 2 Deacylation of peracetylated pyranoses catalyzed by PSL in
different solvents containing 10% v/v of water
mannose were not recognized, probably because the acetoxy
group at carbon 2 is inverted with respect to gluco- and galacto-
pyranoses, which can be accepted as substrate by this enzyme.
Also variation at the C-4 position of pyranoses somehow
affected the activity of PSL, although with a minor inuence
compared with changes at C-2. Accordingly, N-acetylated
pyranosides were less or poorly recognized by PSL compared
with the corresponding substrates bearing in C-2 an acetoxy
group and peracetylated galactosamine 5b provided much
worst results compared with glucosamine 4b.
Entry
Substrate
Solvent
Time (h)
Producta (conv. %)
1
2
3
4
5
6
7
8
1a
2b
2a
3b
3a
4b
4a
5b
5a
1a
2b
2a
4b
4a
S1
S1
S1
S1
S1
S1
S1
S1
S1
S2
S2
S2
S2
S2
24
24
24
24
24
24
24
24
24
24
24
24
24
24
1a (ND)b
2a (96)
2a (ND)
3a (ND)
3a (ND)
4a (71)
4a (ND)
5a (15)
5a (ND)
1a (ND)
2a (40)
2a (ND)
4a (39)
4a (ND)
These results conrmed how the use of this green
commercial solvent can be considered a new and promising
approach for developing sustainable bio-processes for
substrates characterized by a very poor water solubility.37
9
10
11
12
13
14
a
Determined by HPLC: Asahipak column NH2P50-4E (4.6 mm ꢁ 250
Alcoholysis of peracetylated substrates using P. stutzeri lipase
mm); ELSD detector; mobile phase CH3CN–H2O, 80 : 20 (v/v), ow
rate 0.8 mL minꢀ1
.
ND: not detected (<1%).
b
In order to study the alcoholysis of peracetylated substrates
catalyzed by PSL, 1b and 2b, which provided the best results in
the hydrolytic reaction, were employed as substrates. Not only
common alcohols such propanol and 1-butanol were used, but
also solvents S2, S3, S5 and S7, bearing a free OH group (Fig. 1),
were included in the study.
As can be seen in Table 2 very high conversions, similar to
those achieved for substrate 1b, were obtained in the case of
compound 2b when S1 was employed as solvent (entry 2). The
lipase displayed very high selectivity and it exclusively
hydrolyzed the anomeric position. However, although this
enzyme displayed also activity working in S2, the employ of this
solvent led to much lower yields and in some cases no activity
was observed. No conversion was detected if peracetylated
mannoses 3a and 3b were used as substrates. In fact, formation
of product 3a in moderate yields was previously described using
immobilized Lecitase,19 but this product was not obtained with
PSL. In contrast, good results were obtained with PSL when the
b anomer of N-acetylglucosamine (4b) was considered (entry 6).
However, in this last case, the lipase displayed a substantial
lower performance compared with the homologous 2b (71% of
yield was obtained in 24 h), attributed to the presence of the
acetamide group in the position C-2, according to the results
previously obtained with other enzymes.8,14,19 The activity of this
enzyme was further reduced in the hydrolysis of peracetylated
galactosamine 5b (entry 8) and resulted completely inactive in
the hydrolysis of both anomers of the peracetylated lactose
(result not shown).
On the other hand, it is worth of mention that no production
of monodeacylated product was detected when peracetylated a-
saccharides were employed as substrates. Thus, for the
substrates recognized by PSL (substrates 1, 2 and 4), this lipase
is able to discriminate between the a and the b isomer, being
possible to resolve a mixture of both isomers by a PSL-catalyzed
regio- and stereoselective deacylation reaction.
Based on these results, we suspected that the PSL presented
an active site suitable to accept only small molecules such as
monosaccharides (b-D-lactose was not recognized), with a regio-
and stereo-specicity of the enzyme towards the anomeric
position. Accordingly, any variation at C-2 position strongly
inuenced the activity of this enzyme. In fact, both anomers of
Results reported in Table 3 show that most of the alcohols
tested may be successfully employed as nucleophiles for the
alcoholysis of peracetylated 1b and 2b, except S6 and S7. The
yields afforded with most of alcohols range from 82 to 99%,
which means that the lipase can efficiently use them as accep-
tors for the transfer of the anomeric acetyl group. Due to the
broad variety of alcohols used PSL resistance to different
polarities of solvents was proven. In these sense, solvents
employed in these assays presented log P values ranging from
ꢀ0.60 of S5 up to 1.42 of S2 (Table S2 in ESI†). Thus, the null
activity exhibited by the enzyme in the presence of S6 and S7
may be related to an structural phenomena, where the access of
these alcohols to the active site of the enzyme may be limited by
the branched structure of the two groups (tert-butyl and
i-propyl) present in these compounds.
Table 3 Alcoholysis of peracetylated monosaccharides catalyzed by
P. stutzeri lipase in presence of anhydrous alcohols
Entry
Substrate
Alcohol
Time (h)
Producta (conv. %)
1
2
3
4
5
6
7
8
1b
1b
1b
1b
1b
1b
1b
2b
2b
2b
2b
2-Propanol
1-Butanol
S2
S3
S5
S6
S7
24
24
48
48
48
48
48
24
48
48
48
1a (82)
1a (87)
1a (99)
1a (71)
1a (84)
NDb
NDb
1-Butanol
2a (84)
2a (97)
2a (88)
2a (97)
9
10
11
S2
S3
S5
a
Determined by HPLC: Asahipak column NH2P50-4E (4.6 mm ꢁ 250
mm); ELSD detector; mobile phase CH3CN–H2O, 80 : 20 (v/v), ow
rate 0.8 mL minꢀ1
.
ND: not detected (<1%).
b
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It is important to note that the results obtained in the study
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of different solvents showed that the best performances can be
achieved using the solvent S2. This solvent provided the best
results in the alcoholysis of substrate 1b. In particular, PSL
displayed a high preference to catalyze the reaction in the
presence of solvent S2, compared with other solvents derived
from glycerol. The behaviour of the lipase may be attributed to
some specic interaction between the enzyme and the solvent
as it has been previously described for other enzymes.38–40
Consequently, S2 can be considered a promising new green
solvent suitable to be used for developing enzymatic bio-
processes.
Scale up process
Fig. 3 Top view of the cavity of lipase from P. stutzeri with 1b mole-
cule, with the lids on the sides. In blue, large pocket; small pocket in
red. Behind the oxianionic pocket is placed and the group of the
anomeric carbon of 1b.
An important point that must be evaluated aer the synthesis of
an interesting building block is the feasibility of scaling up the
process (hydrolysis and alcoholysis). In this respect, an advan-
tage of solvents S1 and S2 is that under stirring conditions an
emulsion is created between biosolvent and aqueous buffer,
then aer reaction these biosolvents can be separated from
reaction media by centrifugation. Moreover, carbohydrate
compounds in the reaction media are not soluble in the bio-
solvent phase and remains in the aqueous phase. Centrifuga-
tion becomes a very useful tool for the isolation of the
biosolvent from the reaction media, allowing its reuse in further
reactions.
For substrate 1b preparative processes were performed
increasing the reaction volume and using solvent S1 for
hydrolysis and S2 for alcoholysis. In both cases a complete
conversion of the substrate into product was obtained (95%).
With this optimized separation procedure in hand, at the
end of the reaction, the biosolvent (S1 or S2) were recycled and
reused and the product was easily puried by column chro-
matography using dichloromethane–methanol (95 : 5) as eluent
(90% yield). Being the reaction almost complete (97–98%
conversion at 24 h), the product recovered was pure at 95%. This
process was repeated 3 times without losing catalytic action.
The comparison of the enzymatic process with the chemical
reaction reported for the synthesis of product 1a33 showed that
the enzymatic approach gives the best performances in terms of
yields and environmental sustainability. In fact, the chemical
process was performed in DMF using hydrazine in acetic acid,
and provided 65% of yield (Scheme 2). Furthermore, for product
isolation, purication was required, while the enzymatic reac-
tion provided a product with a good purity by a simple work-up.
available model of Pseudomonas mendocina (99% identity with
PSL).39,41 The model has been rened using the AMBER force
eld 03 and a steepest descent algorithm to minimize the
energy function associated with the eld for 2000 steps. Various
parameters have been validated as Ramachandran angles and
hydrophobicity proles of residues, being all comparable to
native structures crystallized. For the docking program was
used Autodock 4.0.42 In the proposed model, the enzyme has
two pockets, small and large, the latter being clearly open to the
solvent. The active site is protected by two lids, which are
opened and in hydrophobic media tend to close in the presence
of water (Fig. 3). In the case of the peracetylated derivatives, the
substrate ts into the active site by size and shape, occupying
the small pocket and exposing the rest of acetyl groups (large
pocket), while the acetyl group at the anomeric position is
arranged inferiorly to the oxoanionic pocket within striking
distance of the catalytic serine (Ser-109). This arrangement is
facilitated by a hydrogen bond, established between the Tyr-54
and the acetyl carbonyl of C-2 (Fig. 4). This residue has been
shown to be crucial for enzyme activity.41
Molecular modelling and docking studies for P. stutzeri lipase
As described in material and methods, a molecular modelling
study was carried out by homology of PSL and the already
Fig. 4 Peracetylated galactose in the active site as proposed binding
mode. Tyr-54 to the right and back Ser-109.
Scheme 2 Chemical deprotection of 1b.33
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any toxic reagent and solvents required in the classical chemical
process.
Accordingly, PSL can be proposed as a useful biocatalyst for
the preparation of acetylated monosaccharides with free
hydroxyl group at the C-1 position. The bio-process can be
performed either by hydrolysis or alcoholysis, using green
solvents as reaction medium; besides, the process of deacyla-
tion may be scaled up with high efficiency and under green
conditions respect to traditional chemical deprotection of the
anomeric position.
The PSL catalyzed also alcoholysis in presence of several
alcohols. In this case the best results were obtained using
solvent S2 and reactions can be affected by the presence of
voluminous groups such t-butyl or iso-propyl residues as part of
the side chains of secondary alcohol. By using the 3D structure
of this lipase we have demonstrated that this enzyme is highly
selective for b-peracetylated monosaccharides without activity
towards the a-anomers. Thus, the regioselective hydrolysis of
peracetylated monosaccharides catalyzed by PSL can be poten-
tially employed as a facile procedure for the bio-separation of
the two anomers too. The 3D structure of the active site also
explains the reduced or the lack of activity observed in the case
of peracetylated mannopyranoses and 2-acetaminopyranoses.
In fact, any change in C-2 strongly inuenced the enzyme
activity at the close anomeric position, as consequence of a
change of the interaction of the group in C-2 with the Tyr-54.
Also this molecular modelling and docking studies
explained the alcoholysis results obtained with S2 solvent.
Small molecules of alcohols are able to enter to the active site of
the enzyme to react with the Ser-109, while the presence of iso-
propyl and t-butyl residues as part of the side chains of the
alcohol avoids them from going into the pockets of the enzyme.
Thus, we could demonstrate that the lipase PSL efficiently
catalyzes the hydrolysis or alcoholysis of peracetylated of b-D-
galactose, b-D-glucosamine and b-D-glucose in the presence of
green solvents, allowing for almost quantitative conversion in
S1 and S2. These solvents are considered safe and should be
appropriate for the synthesis of building blocks that are very
important in the synthesis of oligosaccharides of biological
relevance. Furthermore, reaction scaling up is feasible without
losing catalytic action.
Fig. 5 Top view of the cavity of lipase from P. stutzeri with S6 in the
enzyme active center. The solvent has a t-butyl group that avoids the
approach of the catalytic residue Ser-109 and therefore can not allow
the nucleophilic attack of the alcoholysis reaction.
As we expected, this model could also explain why yields of
N-acetylated derivatives (GalNAc or GlcNAc) and mannose (axial
C-2) are so low. In the N-acetylated sugars, substrate binding
could be hampered by the required planarity demanded by
amide bond, while ethers may establish a H-bonding accepting
the hydrogen from Tyr-54. In the case of mannose, the axial
arrangement of the hydroxyl on C-2 prevents the substrate of
being close enough to establish that interaction. The solvent in
this model 3 interacts directly with acetyl groups, whereby their
effect would be derived precisely from this fact. Depending on
the characteristics of the solvent, these groups would be stabi-
lized by exposure, or otherwise be forced to nonproductive
binding modes to try repelling contact. For the alcoholysis, the
proposed hypothesis was conrmed. The alcohols which are
recognized by the enzyme in these reactions are: ethanol, 1-
butanol, S2, S3 and S5; they are able to enter the active site of the
enzyme to react with the Ser-109, while the two alcohols not
recognized by the enzyme (S6 and S7) have a steric effect caused
by the presence of chains of type i-propyl (S7) and t-butyl (S6),
which avoids them from going into the pockets of the enzyme
(Fig. 5).
Conclusions
Experimental
General
In this work we describe an efficient enzymatic approach for
preparing acetylated pyranosides (commercially available or
easily obtained by chemical acetylation) bearing a free hydroxyl Peracetylated monosaccharides and disaccharides (e.g. b-D-glu-
group only at the anomeric position. These intermediates are copyranose, b-D-galactopyranose, b-D-lactose, etc, Scheme 1),
largely used for preparing the sugar donors currently used in solvents (ethanol, tetrahydrofuran THF and 2-methyltetrahy-
chemical glycosylation and are currently prepared by using drofuran 2-MeTHF) and p-nitrophenyl palmitate (pNPP) were
hazardous reagents and toxic solvents.
purchased from Sigma Aldrich. Solvent S1, (Fig. 1) was a gi
The results reported in the present work provide two major from COGNIS IP Management GmbH now part of BASF.
advantages: rst, we found a lipase that cleaves with high Solvents S2, S3, S4, S5, S6 and S7 (Fig. 1) were a generous gi
26
´
regioselectivity the acetyl group at the anomeric position of from Prof. Ignacio Garcıa, (Universidad de Navarra, Spain;
different peracetylated b-monosaccharides and, secondly, we solvents features are shown in Table S2 see ESI†) Commercially
performed this regioselective deacylation under environmental available lipases from Pseudomonas stutzeri (PSL) and Alcali-
friendly conditions, by using green solvents suitable to achieve genes sp. (QLC) were purchased from Meyto Sangyo (Japan).
high substrate concentration (30 g Lꢀ1) and avoiding the use of CAL-B was purchased from Sigma Aldrich. HPLC Jasco with
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evaporative light scattering detector using NH2P50-4E amino
column (Asahipak, Japan) eluted at 0.8 mL minꢀ1, 80% aceto-
nitrile and 20% water was used. NMR spectra were recorded in
CDCl3 (d ¼ ppm) on a Bruker AV. 250 MHz and a Bruker AV III.
400 MHz.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranose (4a).
Colorless oil, 65% yield. 1H NMR (400 MHz, CDCl3) d (ppm): 6.00
(d, J ¼ 9.5 Hz, 1H, NH), 5.33–5.26 (m, 2H), 5.14 (t, 1H, J ¼ 9.4 Hz),
4.65 (d, 1H, J ¼ 8.5 Hz, H-1 b-anomer), 4.32–4.27 (m, 1H), 4.23–4.20
(m, 2H), 4.16–4.10 (m, 1H), 2.11 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H),
1.98 (s, 3H). 13C NMR (101 MHz, CDCl3) d (ppm): 172.42, 171.93,
171.47, 170.39, 92.48, 71.89, 69.18, 68.46, 63.04, 53.26, 24.09, 21.07,
21.58. Anal. calcd for C14H21NO9: C, 48.41%; H, 6.09%; N, 4.03%;
found: C, 48.29; H, 6.10; N, 4.04%.
Enzyme activity assay
The activities of the lipases were analyzed spectrophotometri-
cally measuring the increment in the absorption at 410 nm
produced by the released of p-nitrophenol in the hydrolysis of 1
mM p-nitrophenyl palmitate (pNPP) in 50 mM sodium phos-
phate buffer at pH 7 and 37 ꢂC. To initiate the reaction 10 mL of
the lipase solution (1.4 mg mLꢀ1 in 50 mM sodium phosphate
buffer pH 7) were added to 2.5 mL of substrate solution. The
variation of the absorbance was monitored for 3 minutes. Each
experimental assay was run at least three times with standard
deviation under 5% of the samples average. An activity unit was
dened as the amount of enzyme necessary to hydrolyze 1 mmol
of pNPP per minute under the conditions described above.
The protein concentration was determined using Bradford
method.43 The protein calibration curve was obtained
using BSA.
Alcoholysis of peracetylated substrates using P. stutzeri lipase
Taking into account the catalytic behaviour of PSL toward per-
acetylated monosaccharides, 1b and 2b were selected for alco-
holysis reactions. 15.0 mg of each substrate were dissolved in
500 mL of dry alcohol (1-butanol, S2, S3, S5, S6 and S7). 10.0 mg
of PSL17 and molecular sieves (50.0 mg) were added to the
mixture and the reactions were shaked at 30 ꢂC during 48 h.
Alcoholysis reactions were monitorized by HPLC. Aer
complete reaction, products were puried by column chroma-
tography using dichloromethane–methanol (95 : 5) as eluent.
Solvent was evaporated and puried products were analysed by
1H-NMR and 13C-NMR. Structures assignment was performed
by means of 2D-COSY, HSQC experiments and HMBC. NMR
spectra were similar to those reported above and were concor-
dant with literature.6,8
Hydrolysis of peracetylated substrates using P. stutzeri lipase
Standard assay was performed as follows: 15.0 mg of the per-
acetylated sugar were solved in 500 mL of solvent containing 50
mL of distilled water. The reaction was started by the addition of
10.0 mg of PSL17 and the reaction progress was monitorized by
HPLC. For identication of products, aer complete reaction,
the product was puried by column chromatography using
dichloromethane–methanol (95 : 5) as eluent. Solvent was dried
and puried products were analysed by 1H-NMR and 13C-NMR.
Structures assignment was performed by means of 2D-COSY,
HSQC experiments and HMBC. The NMR analytical data were
corresponding to those reported in literature.6,8,44
Preparative hydrolysis and alcoholysis
Substrate 1b (2.4 g, 6.1 mmol) was dissolved in 80 mL of S1 with
10% of distilled water (for hydrolysis) and in 80 mL of anhy-
drous S2 containing 10% m/v of molecular sieves (for
alcoholysis) by stirring during 24 hour at 30 ꢂC. The biocatalyst
(20 mg mLꢀ1) was then added to the reaction mixture under
stirring. Aliquots (100 mL) were withdrawn at different intervals,
ultra-ltered, and the progress of the reaction was checked by
HPLC analysis. Aer complete reaction the biosolvent (S1 or S2)
was recycled and reused three times by centrifugation and the
product 1a was puried by column chromatography using
dichloromethane–methanol (95 : 5) as eluent. Solvent was dried
and puried products were analyzed by 1H-NMR and 13C-NMR.
Structures assignment was performed by means of 2D-COSY,
HSQC experiments and HMBC. NMR spectra were similar to
those data previously reported and were concordant with liter-
ature.6,8,44 This process was repeated 3 times without losing
catalytic action.
2,3,4,6-Tetra-O-acetyl-a-D-galactopyranose (1a). Colorless oil,
1
90% yield (mixture of anomers). H NMR (400 MHz, CDCl3) d
(ppm): 5.52 (bd, J ¼ 3.4 Hz, 1H, H-1 a-anomer), 5.48 (dd, 1H, J ¼
1.3 Hz), 5.41 (dd, 1H, J ¼ 3.5 Hz), 5.18 (dd, 1H, J ¼ 3.4 Hz), 4.48
(t, 1H, J ¼ 6.7 Hz), 4.11–4.09 (m, 2H), 2.10 (s, 3H), 2.06 (s, 3H),
2.04 (s, 3H), 2.00 (s, 3H). 13C NMR (101 MHz, CDCl3) d (ppm):
171.5, 171.3, 171.2, 171.0, 96.9 (C-1 b-anomer), 91.0 (C-1 a-
anomer), 71.9, 71.3, 69.2, 68.1, 62.7, 21.9, 21.7, 21.6, 21.5. Anal.
calcd for C14H20O10: C, 48.28%; H, 5.79%; found: C, 48.09; H,
5.80%. [a]2D0: +54.6 (c 1.98, CHCl3).
Molecular modelling and docking
2,3,4,6-Tetra-O-acetyl-a-D-glucopyranose (2a). Colorless oil,
1
93% yield (mixture of anomers). H NMR (400 MHz, CDCl3) d In order to explain results obtained by the hydrolysis and
(ppm): 5.54 (t, J ¼ 9.7 Hz, 1H), 5.46 (d, 1H, J ¼ 3.5 Hz, H-1 a- alcoholysis of peracetylated sugars with PSL, we decided to
anomer), 5.09 (t, 1H, J ¼ 9.8 Hz), 4.91 (q, 1H, J ¼ 3.0 Hz), 4.74 (d, develop a study of molecular modelling and docking. The
1H, J ¼ 8.5 Hz, b-anomer), 4.29–4.12 (m, 3H), 2.10 (s, 3H), 2.09 model was constructed by homology using as input the three-
(s, 3H), 2.04 (s, 3H), 2.02 (s, 3H). 13C NMR (101 MHz, CDCl3) d dimensional structure of the lipase from Pseudomonas mendo-
(ppm): 171.8, 171.7, 171.1, 170.6, 96.4 (C-1 b-anomer), 91.0 (C-1 cina (99% homology).41 The model has been rened using the
a-anomer), 72.0, 70.8, 69.4, 68.1, 62.9, 21.6, 21.5. Anal. calcd for AMBER force eld 03 and a steepest descent algorithm to
C
14H20O10: C, 48.28%; H, 5.79%; found: C, 48.12; H, 5.81%. minimize the energy function associated with the eld for 2000
[a]2D0: +46.9 (c 2.49, CHCl3).
steps. Various parameters have been validated as
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Ramachandran angles and hydrophobicity proles of residues, 19 G. Fernandez-Lorente, M. Filice, M. Terreni, J. M. Guisan,
being all comparable to crystallized native structures. For the
docking program was used Autodock 4.0.42 In the proposed
R. Fernandez-Lafuente and J. M. Palomo, J. Mol. Catal. B:
Enzym., 2008, 51, 110–117.
model, the enzyme has two pockets, small and large, the latter 20 O. Miyawaki and M. Tatsuno, J. Biosci. Bioeng., 2008, 105, 61–
being clearly open to the solvent. The active site is protected by 64.
two lids, which are opened and in hydrophobic media tend to 21 P. T. Anastas and J. C. Warner, Green Chemistry: Theory and
close in the presence of water.
Practice, New York, 1998.
22 P. E. Anastas and N. Eghbali, Chem. Soc. Rev., 2010, 39, 301–
312.
23 R. A. A. I. H. U. Sheldon, Green Chemistry and Catalysis,
Wiley-VCH, Weinheim, 2007.
Acknowledgements
This work was supported by a research project from the Spanish
´
˜
MICINN (Ministerio de Ciencia e Innovacion de Espana
´
´
´
24 M. Perez-Sanchez, M. Sandoval and M. J. Hernaiz,
CTQ2012-32042).
Tetrahedron, 2012, 68, 2141–2145.
25 M. Perez-Sanchez, M. Sandoval, M. J. Hernaiz and
´
´
P. Domınguez de Marıa, Curr. Org. Chem., 2013, 17, 1188–
1199.
Notes and references
1 M. E. C. Caines, H. Zhu, M. Vuckovic, L. M. Willis,
S. G. Withers, W. W. Wakarchuk and N. C. J. Strynadka, J.
Biol. Chem., 2008, 283, 31279–31283.
2 H. Shirato, S. Ogawa, H. Ito, T. Sato, A. Kameyama,
H. Narimatsu, Z. Xiaofan, T. Miyamura, T. Wakita, K. Ishii
and N. Takeda, J. Virol., 2008, 82, 10756–10767.
3 S. Nagaraj, K. Gupta, V. Pisarev, L. Kinarsky, S. Sherman,
L. Kang, D. L. Herber, J. Schneck and D. I. Gabrilovich,
Nat. Med., 2007, 13, 828–835.
4 K. E. Niederer, D. K. Morrow, J. L. Gettings, M. Irick,
A. Krawiecki and J. L. Brewster, Cell. Signalling, 2005, 17,
177–186.
5 H. Yagi, M. Yanagisawa, Y. Suzuki, Y. Nakatani, T. Ariga,
K. Kato and R. K. Yu, J. Biol. Chem., 2010, 285, 37293–37301.
6 M. Filice, J. M. Guisan, M. Terreni and J. M. Palomo, Nat.
Protoc., 2012, 7, 1783–1796.
´
´
´
´
26 J. I. Garcıa, H. Garcıa-Marın, J. A. Mayoral and P. Perez, Green
Chem., 2010, 12, 426–434.
27 A. Wolfson, C. Dlugy and Y. Shotland, Environ. Chem. Lett.,
2007, 5, 67–71.
28 P. Lozano, Green Chem., 2010, 12, 555–569.
29 H. R. Hobbs and N. R. Thomas, Chem. Rev., 2007, 107, 2786–
2820.
30 R. A. Sheldon, Green Chem., 2005, 7, 267–278.
31 X. Tao, Curr. Opin. Chem. Biol., 2009, 13, 1–18.
32 M. Alcalde, M. Ferrer, F. J. Plou and A. Ballesteros, Trends
Biotechnol., 2006, 24, 281–287.
33 H. Cheng, X. Cao, M. Xian, L. Fang, T. B. Cai, J. J. Ji,
J. B. Tunac, D. Sun and P. G. Wang, J. Med. Chem., 2004,
48, 645–652.
´
´
´
34 M. Perez-Sanchez, M. Sandoval, A. Cortes-Cabrera,
´
´
´
H. Garcıa-Marın, J. V. Sinisterra, J. I. Garcıa and
M. J. Hernaiz, Green Chem., 2011, 13, 2810–2817.
35 D. A. Blake, H. F. Cascorbi, R. S. Rozman and F. J. Meyer,
Toxicol. Appl. Pharmacol., 1969, 15, 83.
7 T. Bavaro, M. Filice, P. Bonomi, Q. Abu alassal, G. Speranza,
J. M. Guisan and M. Terreni, Eur. J. Org. Chem., 2011, 6181–
6185.
8 M. Filice, R. Fernandez-Lafuente, M. Terreni, J. M. Guisan
and J. M. Palomo, J. Mol. Catal. B: Enzym., 2007, 49, 12–17.
36 M. J. Murphy, D. A. Dunbar and L. S. Kaminsky, Toxicol. Appl.
Pharmacol., 1983, 71, 84–92.
˜
9 S. Inigo, M. T. Porro, J. M. Montserrat, L. E. Iglesias and
´
37 P. Lozano, E. Garcıa-Verdugo, J. M. Bernal, D. F. Izquierdo,
A. M. Iribarren, J. Mol. Catal. B: Enzym., 2005, 35, 70–73.
10 U. T. Bornscheuer, Curr. Opin. Biotechnol., 2002, 13, 543–547.
11 V. Gotor-Fernandez, R. Brieva and V. Gotor, J. Mol. Catal. B:
Enzym., 2006, 40, 111–120.
´
´
M. I. Burguete, G. Sanchez-Gomez and S. V. Luis,
ChemSuschem, 2012, 5, 790–798.
38 M. Z. Kamal, P. Yedavalli, M. V. Deshmuck and N. M. Rao,
Protein Sci., 2013, 22, 904–915.
12 E. Busto, V. Gotor-Fernandez and V. Gotor, Chem. Soc. Rev.,
2010, 39, 4504–4523.
13 M. Kapoor and M. N. Gupta, Process Biochem., 2012, 47, 555–
569.
14 M. Filice, D. Ubiali, R. Fernandez-Lafuente, G. Fernandez-
Lorente, J. M. Guisan, J. M. Palomo and M. Terreni, J. Mol.
Catal. B: Enzym., 2008, 52–53, 106–112.
˜
´
39 M. Sandoval, C. Civera, J. Trevino, E. Ferreras, A. Cortes,
M. Vaultier, J. Berenguer, P. Lozano and M. J. Hernaiz, RSC
Adv., 2012, 2012, 6306–6314.
40 S. J. Nara, J. R. Harjani and M. M. Salunkhe, Tetrahedron
Lett., 2002, 43, 2979–2982.
41 A. Maraite, P. Hoyos, J. D. Carballeira, A. C. Cabrera,
´
´
M. B. Ansorge-Schumacher and A. R. Alcantara, J. Mol.
15 A. M. Gumel, M. S. M. Annuar, T. Heidelberg and Y. Chisti,
Process Biochem., 2011, 46, 2079–2090.
16 A. Wolfson, A. Atyya, C. Dlugy and D. Tavor, Bioprocess
Biosyst. Eng., 2010, 33, 363–366.
Catal. B: Enzym., 2013, 87, 88–98.
42 G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner,
R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput.
Chem., 2009, 30, 2785–2791.
43 M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
44 T. B. Cai, D. Lu, X. Tang, Y. Zhang, M. Landerholm and
P. G. Wang, J. Org. Chem., 2005, 70, 3518–3524.
˜
17 E. D. Gudino, A. M. Iribarren and L. E. Iglesias, Tetrahedron:
Asymmetry, 2009, 20, 1813–1816.
18 J. M. Palomo, M. Filice, R. Fernandez-Lafuente, M. Terreni
and J. M. Guisan, Adv. Synth. Catal., 2007, 349, 1969–1976.
55502 | RSC Adv., 2014, 4, 55495–55502
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