protocol
Regioselective monodeprotection of peracetylated
carbohydrates
Marco Filice1, Jose M Guisan1, Marco Terreni2 & Jose M Palomo1
Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Madrid, Spain. 2Dipartimento di Scienze del Farmaco, Università di Pavia, Pavia, Italy. Correspondence
should be addressed to J.M.P. ([email protected]).
1
© 2012 Nature America, Inc. All rights reserved.
Published online 6 September 2012; doi:10.1038/nprot.2012.098
This protocol describes the regioselective deprotection of single hydroxyls in peracetylated monosaccharides and disaccharides
by enzymatic or chemoenzymatic strategies. The introduction of a one-pot enzymatic step by using immobilized biocatalysts
obviates the requirement to carry out tedious workups and time-consuming purifications. By using this straightforward protocol,
different per-O-acetylated glycopyranosides (mono- or disaccharides, 1-substituted or glycals) can be transformed into a whole
set of differentially monodeprotected 1-alcohols, 3-alcohols, 4-alcohols and 6-alcohols in high yields. These tailor-made glycosyl
acceptors can then be used for stereoselective glycosylation for oligosaccharide and glycoderivative synthesis. They have been
successfully used as building blocks to synthesize tailor-made di- and trisaccharides involved in the structure of lacto-N-neotetraose and precursors of the tumor-associated carbohydrate antigen T and the antitumoral drug peracetylated b-naphtyllactosamine. We are able to prepare a purified monoprotected carbohydrate in between 1 and 4 d. With this protocol, the small
library of monodeprotected products can be synthesized in 1–2 weeks.
INTRODUCTION
Carbohydrates occur naturally as oligo- and polysaccharides and
can also be coupled to, for example, peptides and lipids. They have
key roles in a broad range of biological processes1–5.
In many cases, the biological activity of natural products derives
from the sugar moieties. Changes in the sugar structures can affect
the biological properties of the parent compounds6,7. Furthermore,
functional properties of drugs such as solubility, pharmacokinetics
or pharmacodynamics can be enhanced by the incorporation of
carbohydrate moieties into the molecules8–10.
Methods for synthesizing these molecules have, therefore,
acquired immense importance, because it is not easy to isolate sufficient amounts of acceptable pure product from natural sources.
Their synthesis is, however, difficult, involving multistep protection, deprotection, activation and glycosylation steps, because they
are complex molecules with multiple chiral centers.
Here, we describe a straightforward protocol for one-pot regioselective synthesis of diversely monodeprotected mono- and
disaccharide building blocks from fully protected carbohydrates
catalyzed by immobilized enzymes11–22 (Fig. 1).
In fact, via an enzyme-catalyzed reaction, the desired building
block bearing a single strategically positioned free hydroxyl group
(a nucleophilic acceptor) can be accessed easily after workup and
simple purification. This field has always been concerned with two
major challenges, regioselective protection of individual hydroxyls and stereoselective glycosylation23–30. Regioselective hydroxyl
protection usually involves multistep protocols and tedious purifications, because all the secondary hydroxyls have comparable
reactivity. In the preparation of glycoderivatives, along with the
stereoselectivity, it is also essential to control the regioselectivity
of glycosylation so that only a specific hydroxyl group is coupled
with the donor sugar.
A traditional chemical approach for carbohydrate synthesis
involves a prior protection of a hexose at the anomeric carbon
(C1) to provide the corresponding hexopyranoside tetraol via the
formation of cyclic acetal. Transformation of the tetraol into either
the fully protected monosaccharide or the individual alcohols with
a free hydroxyl at C3, C4 or C6 is frequently difficult. Although
offering diversity (regioselectivity, orthogonality, nature of protecting groups, reactivity tuning and others), chemical approaches
also present severe drawbacks: (i) an independent and multistep
protection-deprotection sequence is needed to prepare each compound, (ii) a tedious workup is often used in each synthetic step,
(iii) a time-consuming purification is required to separate different
regioisomers and (iv) low yield of the expected product is sometimes obtained owing to the poor regioselectivity31. An enzymatic
process overcomes these drawbacks and is therefore a good alternative approach32–35. The reaction in this protocol is a one-step
deprotection of a fully protected carbohydrate. It is an efficient,
high-yielding process with high regioselectivity to hydrolyze a
unique position.
R3
OAc
AcO
O
X
OH
R2
Biocatalyst, RT
buffer:acetonitrile (80:20)
pH 5
for 20, 22, 31, 36
1-OH
60–63
Biocatalyst, RT
buffer:acetonitrile (80:20) R 3
pH 5
AcO
for 20–28, 30–35, 37–38
R3
OAc
AcO
O
X
R
2
OH
O
X
R2
R1
6-OH
39–55
R1
20–38
R = α/βOAc, βOMe, βOPh, βOpNO2Ph, βOBu, βSisoprop or H
2
3
R = OAc, NHAc or H; R = ax/eq OAc or eqGal4OAc; X = single or double
1
HO
OAc
AcO
O
X
R
2
R1
4-OH
64–72
1
R = α/βOAc or H
2
R = OAc, NHAc or H
X = single or double
1. Biocatalyst, RT
1. Biocatalyst, RT
buffer:acetonitrile (80:20)
buffer:acetonitrile (80:20)
pH 5
pH 5
2. Catalyst filtration
2. Catalyst filtration
3. pH 5 to 8.5, 4 °C 2 h
3. pH 5 to 9.5, 4 °C 2 h
for 20–23, 30–33, 35
for 20–23, 31–33
or
R3
OAc
HO
O
X
R2
R1
3-OH
57–59, 73–79
1
R = α/βOAc or H
Biocatalyst, RT
2
buffer:acetonitrile (80:20) R = OAc, NHAc or H
3
R = ax/eq OAc or eq Gal
pH 5
for 30, 35, 38
X = single or double
Figure 1 | General scheme of the regioselective monodeprotection of per-Oacetylated carbohydrates. RT, room temperature.
nature protocols | VOL.7 NO.10 | 2012 | 1783
protocol
HN
OH
Lipase immobilized
on octyl-Sepharose
OH
Lipase immobilized
on CNBr-Sepharose
Hydrophobic area
© 2012 Nature America, Inc. All rights reserved.
Figure 2 | Immobilized enzymes scheme.
acetylation of glucopyranoses (1, 3) and galactopyranoses
(12, 14) and 1-substituted glycopyranosides (6, 15, 18) using
acetic anhydride and pyridine (Fig. 3) to afford the corresponding
monosaccharides 20, 22, 25, 31, 33, 34 and 37 in near-quantitative
yields (Table 1 and Supplementary Table 1). The corresponding
per-O-acetylated β-anomers glycopyranoses (21, 23, 32), glycopyranosides (24, 26–29) and glycals (30, 35, 38) were available from
commercial sources.
The reaction conditions and the results of their differential
one-pot monodeprotection, generating a vast array of building
blocks, are summarized in Figure 1. Our general protocols involved
(i) regioselective deprotection of per-O-acetylated monosaccharides, disaccharides and glycals catalyzed by immobilized lipases
to yield 1-OH, 6-OH or 3-OH monodeprotected products; and
(ii) acyl chemical migration of the acetyl group from O4 to O6 to
provide 4-OH or from O3 to O6 to provide 3-OH monodeprotected products in α- and β-monosaccharides and glycals.
To initialize the regioselective deprotection, we added the selected
enzyme preparation to an acidic buffered solution of peracetylated
saccharide (monosaccharide, disaccharide or glycal; Fig. 4). We performed the reaction in a pH-Stat to continuously maintain the
pH at 5, with the aim of preventing the chemical acyl migration
catalyzed by neutral or basic pH that can occur after the deprotection of saccharides on primary positions.
When we completed the regioselective hydrolysis, as monitored by thin-layer chromatography (TLC) or high-performance
liquid chromatography (HPLC), we obtained the corresponding
monodeprotected building blocks listed in Tables 2 and 3 and
Supplementary Tables 2 and 3 (6-OH, 3-OH or 1-OH; 39–63) in
high overall yield, generally after extraction (in almost all cases) or
column chromatography (when indicated).
The subsequent step applied in our strategy concerns the production of other building blocks bearing free hydroxyl groups in
positions that were previously not possible to achieve using only
the aforementioned regioselective enzyme catalysis.
With this aim, to obtain the 3-OH and the 4-OH monodeprotected alcohols of the substrates previously deprotected at 6-OH
position by enzymatic hydrolysis, we carried out a controlled
chemical acyl migration17 (Fig. 1). Generally, this reaction has
been described as a secondary undesired reaction where an acyl
group could migrate to an adjacent free hydroxyl group in aqueous
neutral or basic medium. By the fine-tuning of critical parameters
such as pH, time and temperature, we successfully controlled the
shift of the acyl group from the C3 or the C4 position to the 6-OH
For the successful development of a regioselective, orthogonal
and one-pot deprotection scheme, the choice of the protecting
groups was crucial. We opted for acetyl-type esters, which can
be selectively cleaved under the appropriate conditions36,37. Our
strategy was to protect all hydroxyl groups in order to obtain fully
protected building blocks. Acetyl groups can be easily removed by
sodium methoxylate (Zemplén deacetylation) in the final step to
yield free hydroxyl groups.
Hydrolases—in particular, lipases—were chosen for the regioselective deprotection of different peracetylated mono- and disaccharides achieving 1-, 3-, 4- and 6- free hydroxyl groups.
Lipases are very useful enzymes with a high versatility for many
different compounds38,39. A complex mechanism manages their
catalysis on the basis of the conformational changes between a
‘closed’ and an ‘open’ conformation40. This mechanism of action
has permitted the use of different immobilization protocols
(involving different areas of the lipase, rigidity, microenvironments and so on)41,42, causing a strong modulation of the lipase
properties in kinetic resolution of racemic mixtures42 and asymmetric hydrolysis41–43. We tried different immobilized derivatives
of different lipases as catalysts for the regioselective hydrolysis of
peracetylated carbohydrates in aqueous media. By looking for good
catalytic activity and high regioselectivity, we selected the optimum
catalyst for the deprotection of each fully protected saccharide from
a set of enzyme preparations. The two biocatalyst preparations
that performed best in most cases were
(i) enzymes adsorbed on Sepharose coated
with a dense layer of hydrophobic groups Table 1 | Examples in per-O-acetylation of carbohydrates.
(octyl), involving the hydrophobic area
surrounding the active site of the lipase44,
Substrate
R1
R2
R3
X
Producta
TLC
where the open conformation of the
1
αOH
OH
Eq OH Single
20
Hexane:AcOEt 5:5, vol/vol
enzyme is fixed; and (ii) enzymes immobilized on Sepharose activated with CNBr
3
αOH
NHAc Eq OH Single
22
CH2Cl2:MeOH 95:5, vol/vol
by covalent attachment via the most active
6
βOBu
OH
Eq OH Single
25
Hexane:AcOEt 4:6, vol/vol
amino group (usually the N terminus) on
the enzyme surface45 (Fig. 2).
14
αOH
NHAc Ax OH Single
33
CH2Cl2:MeOH 95:5, vol/vol
To test the enzymatic strategy, we selected
a set of different per-O-acetylated mono15
βSPropiso OH
Ax OH Single
34
Hexane:AcOEt 5:5, vol/vol
and disaccharides (20–38) (Table 1 and Ax, axial; Eq, equatorial.
Product was synthesized in > 95% overall yield. See Supplementary Table 1 for the full analytical data.
Supplementary Table 1). We ­ performed
a
1784 | VOL.7 NO.10 | 2012 | nature protocols
protocol
R3
HO
OH
R3
O
R2
Ac2O, DMAP cat, RT
R1
1, 3, 5–10, 12, 14, 15, 17, 18
1
R = α/βOH, βOMe, βOPh,
βOpNO2Ph, βOBu, βSisoprop
2
R = OH or NHAc
3
R = ax/eq OH or eq Gal
AcO
Pyr
OAc
R3
OAc
AcO
O
X
O
R2
R1
20, 22, 24–29, 31, 33, 34, 36, 37
1
R = α/βOAc, βOMe, βOPh,
βOpNO2Ph, βOBu, βSpropiso
2
R = OAc or NHAc
3
R = ax/eq OAc or eq Gal4Ac
Figure 3 | Per-O-acetylation of the different d-glycopyranosides. Pyr, pyridine.
R2
buffer:acetonitrile
(80:20) pH 5
R1
R3
OH
AcO
O
X
R2
R1
39–55
20–28, 30–35, 37–38
R1 = α/βOAc, βOMe, βOPh,
βOpNO2Ph, βOBu, βSpropiso or H
R 2 = OAc or NHAc
R 3 = ax/eq OAc
X = single or double
AcO
AcO
O
AcO
© 2012 Nature America, Inc. All rights reserved.
Biocatalyst, RT
OPh-pNO2
ANL, RT
buffer:acetonitrile
HO
AcO
O
AcO
OPh-pNO2
free group, depending on the steric configuration of the structure
(80:20) pH 5
29
56
(glucosidic or galactosidic)17. Accordingly, we filtered the reacOAc
tion mixture (proceeding from the enzymatic previous step and
OAc
R3
R3
O
Biocatalyst, RT
containing the 6-OH carbohydrate) to remove the heterogeneous
O
AcO
HO
enzyme catalyst. We subsequently cooled the re-collected solution
buffer:acetonitrile
(80:20) pH 5
30, 35, 38
57–59
on ice (0–4 °C) and set the pH to the desired value by fast addition
3
of 6 N NaOH solution, depending on the saccharide structure and
R = ax/eq OAc or eq Gal
the desired outcome of the reaction.
OAc
Generally, the controlled acyl migration reaction, setting the pH
OAc
R3
R3
O
Biocatalyst, rt
O
to 8.5, permits a high yield of 4-OH deprotected alcohol with a
AcO
AcO
small amount of 3-OH building-block formation for 6-OH carbobuffer: acetonitrile
OAc
OH
R2
R2
(80:20) pH 5
hydrates with a glucosidic structure (Table 4 and Supplementary
20, 22, 31, 36
60–63
Table 4). An increase in 3-OH alcohol yield was possible, raising
2
R = OAc or NHAc
3
the pH up to 9.5 (Table 5 and Supplementary Table 5).
R = ax/eq OAc
When we used the 6-OH carbohydrate with a galactosidic strucFigure 4 | Enzymatic monodeprotection of peracetylated carbohydrates.
ture, we obtained an almost equimolar mixture of both regioisomers
(3-OH and 4-OH alcohols), in nearly all cases studied. Probably,
this different behavior can be explained by considering the different
steric hindrance exerted by substituents at C4 than would exist in solubility in water and the reaction rates are low. A lower solubilan axial (galacto) or equatorial (gluco) configuration.
ity in aqueous solution for peracetylated tri- or tetrasaccharides
When we completed the controlled acyl migration (monitored by could require a different optimization using additional cosolvent,
HPLC), we obtained the corresponding monodeprotected building
ionic liquids and so on.
blocks (3-OH, 4-OH, 64–79; Fig. 1 and Supplementary Tables 4
and 5) from moderate to high overall yield after column chroma- Applications of the approach
tography. It is noteworthy that the acetyl ester, the only protecting The enzymatic regioselective deprotection, in some cases comgroup manipulated in the entire protocol, was highly resistant in
bined with a subsequent acyl-migration step (chemo-enzymatic
all the conditions studied.
process), can offer an efficient and convenient protocol for the
In this way, we have efficiently prepared a library of building
preparation of different glycoderivatives. Indeed, some of the
blocks in aqueous media starting from different d-monosaccha- monodeprotected saccharides synthesized by this approach have
rides and disaccharides, and we expect that the protocol is equally been successfully used as building blocks to synthesize different
amenable to other kinds of oligosaccharides (trisaccharides, tet- tailor-made di- and trisaccharides (80–81) involved in the structure
rasaccharides and so on).
of lacto-N-neo-tetraose and one precursor of the tumor-associated
For the success of the one-pot reaction, the experimenter carbohydrate antigen T (ref. 14) (82; Fig. 5). Furthermore, an outshould bear in mind that it is very important to maintain
standing synthesis of the peracetylated β-naphtyl-lactosamine 83
particular acidic reaction conditions (pH 5)
for the proper enzymatic steps. Only Table 2 | Examples in regioselective enzymatic C-6 monodeprotection of per-O-acetylated
when migration is necessary can the pH
glycopyranosides.
can be increased.
In the chemoenzymatic process, 3-OH
Subs
R1
R2
R3
X
Biocatalyst DP Product Yield (%)
and 4-OH monodeprotected products
20
αOAc
OAc Eq OAc Single
CRLa
C-6
39
96
are always produced as a mixture, and it is
necessary to carefully set the temperature
24
βOMe
OAc Eq OAc Single
CRLa
C-6
43
95
and pH conditions in the incubation (at
25
βOBu
OAc Eq OAc Single
ANLb
C-6
44
77c
alkaline pH) steps to achieve the optimal
regioselectivity for each molecule. In some
32
βOAc
OAc Ax OAc Single
LECI/TLLa
C-6
50
95
cases, particularly for the gluco- and galacAx, axial; DP, deprotected position; Eq, equatorial; Subs, substrate.
tosamine compounds (especially for those
Lipase was immobilized on octyl-Sepharose. Lipase was immobilized on CNBr-Sepharose. Purified by column chromatography.
1-substituted with aromatic rings), the See Supplementary Table 2 for the full analytical data.
a
b
c
nature protocols | VOL.7 NO.10 | 2012 | 1785
protocol
(antitumoral drug) was achieved by this
strategy15, reducing the synthetic steps and
increasing the final overall yield compared
with the chemical method46 (Fig. 5).
Therefore, these interesting results suggest that this strategy shows a great potential
for application in the generation of crucial
glycoconjugates, in tailor-made protein glycosylation, in the modification of natural
products and in nanoglycobiology.
Table 3 | Examples in regioselective enzymatic monodeprotection of per-O-acetylated
glycopyranosides.
Subs
R1
R2
R3
X
Biocatalyst
29
βOPhNO2
OAc
Eq OH
Single
ANLa
30
H
H
Eq OAc Double
21
βOAc
OAc
Eq OAc Single
DP
Product
Yield (%)
C-4
56
96
CAL-Ba
C-3
57
99
PFLa
C-1
60
96
DP, deprotected position; Eq, equatorial; Subs, substrate.
a
Lipase was immobilized on octyl-Sepharose. See Supplementary Table 3 for the full analytical data.
© 2012 Nature America, Inc. All rights reserved.
MATERIALS
• Lipase from Candida antarctica (fraction B, CAL-B; Novozymes,
REAGENTS
cat. code Lipozyme CALBL)
• Deionized water (used in all solutions)
• Lipase from Thermomyces lanuginose (TLL; Novozymes,
• d-Glucopyranose (Sigma-Aldrich, cat. no. G8270)
cat. code Lipozyme TL100L)
• d-Galactopyranose (Sigma-Aldrich, cat. no. G0750)
• Lipase from Rhizomucor miehei (RML; Novozymes, cat. code Palatase
• 2-Acetamido-2-deoxy-d-galactopyranose (Sigma-Aldrich, cat. no. A2795)
20000L)
• 2-Acetamido-2-deoxy-d-glucopyranose (Sigma-Aldrich, cat. no. G4875)
• Lipase from Pseudomonas fluorescens (PFL; Sigma-Aldrich, cat. no 534730)
• 1,2,3,4,6-Penta-O-acetyl-β-d-glucopyranose (21; Sigma-Aldrich,
• Lipase from Candida rugosa (CRL; Sigma-Aldrich, cat. no. L1754)
cat. no. 285943)
• Immobilized Acetyl Xilan esterase from Bacillus pumilus (ACS Dobfar)
• 2-Acetamido-2-deoxy-1,3,4,6-penta-O-acetyl-β-d-glucopyranose (23;
• Sodium phosphate monobasic hydrate (NaH2PO4·H2O; Sigma-Aldrich,
Sigma-Aldrich, cat. no. 859990)
cat. no. 71504)
• 1,2,3,4,6-Penta-O-acetyl-β-d-galactopyranose (32; Sigma-Aldrich,
• Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O; Sigma-Aldrich,
cat. no. 134131)
cat. no. 30412)
• 3,4,6-Tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol(triacetyl
• 4-Nitrophenyl butyrate (pNPB; Sigma-Aldrich, cat. no. N9876)
glucal) (30; Sigma-Aldrich, cat. no. T44407)
• Triton X-100 (Sigma-Aldrich, cat. no. X100)
• Tri-O-acetyl-d-galactal (35; Iris Biotech, cat. no. GBB1213)
• Hexadecyltrimethylammonium bromide (CTAB; Sigma-Aldrich,
• 1-O-Methyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside (Sigma-Aldrich,
cat. no. H9151)
cat. no. S963712)
• Octyl-Sepharose (GE Healthcare, cat. no. 17-0946-02)
• 1-O-Butyl-β-d-glucopyranoside (BOC Science, cat. no. 5391-18-4)
• CNBr-Sepharose (GE Healthcare, cat. no. 17-0981-01)
• 1-Thioisopropyl-β-d-galactopyranoside (Alfa Aesar, cat. no. B21149)
• Acetonitrile (99.8%; Sigma-Aldrich, cat. no. 271004)
• 1-O-Phenyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside (Aurora Screening
• Ethyl acetate (Merck, cat. no. 109623) ! CAUTION It is highly flammable and
Library, cat. no. K07.530.224)
irritating to eyes; repeated exposure may cause skin dryness or cracking.
• 1-O-(4-Nitrophenyl)-2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside
• Anhydrous Na2SO4 (Sigma-Aldrich, cat. no. 239313) ! CAUTION Respira(TRC-Canada, cat. no. N505740)
tory protection is not required. Use gloves while handling it. Use equipment
• 2-Acetamido-2-deoxy-1-(4-nitrophenyl)-3,4,6-tri-O-acetyl-β-dfor eye protection. It forms hazardous decomposition products under fire
glucopyranoside (TRC-Canada, cat. no. N502200)
conditions (sulfur and sodium oxides).
• 1-(4-Nitrophenyl)-2,3,4-tri-O-acetyl-β-d-xylopyranoside (BOC Sciences,
• Methanol (EMD, cat. no. MX0485-5) ! CAUTION It is highly flammable and
cat. no. 24624-78-0)
can cause acute toxicity (oral, dermal, inhalation), respiratory sensitization,
• Hexa-O-acetyl-1,5-anhydro-2-deoxy-4-O-β-d-galactopyranosyl-dgerm cell mutagenicity, carcinogenicity, and reproductive toxicity; it is an
arabinohex-1-enitol (hexaacetyl lactal) (38; Iris Biotech, cat. no. GBB1214)
aspiration hazard.
• 2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl-(1→4)-1,2,3,6-tetra-O• DCM (Merck, cat. no. 106050) (dried over Al2O3) ! CAUTION There is limacetyl-β-d-glucopyranoside (lactose; Iris Biotech, cat. no. GBB1150)
ited evidence of a carcinogenic effect; do not breathe vapor, avoid contact
• Acetic anhydride (Merck, cat. no. 822278) ! CAUTION Acetic anhydride is
with skin and eyes, and wear suitable protective clothing and gloves.
flammable; it is harmful by inhalation and if swallowed.
• Silica gel (Merck, cat. no. 1.09385.2500) ! CAUTION Do not breathe dust;
• Pyridine (Sigma-Aldrich, cat. no. 270970) ! CAUTION Pyridine is highly
wear latex gloves.
flammable and reacts violently with water; it is harmful by inhalation, on
contact with skin or if swallowed.
• Triethylamine (Riedel-de Haën, cat. no. 16304)
! CAUTION Triethlyamine is highly flammable; it
Table 4 | Synthesis of 4-hydroxy-tetraacetylated monosaccharides by acyl-chemical
is harmful by inhalation, on contact with skin or
migration from the 6-OH monodeprotected tetraacetylated products.
if swallowed. It causes severe burns; keep it in a
cool place.
Substrate
R1
R2
R3
X
pHa DP
Product Yield (%)
• 4-(Dimethylamino)pyridine (DMAP; SigmaAldrich, cat. no. 107700)
20
αOAc OAc
Eq OAc
Single 8.5 C-4
64
80
• Molecular sieves (3 Å; Sigma-Aldrich, cat. no.
233641) ! CAUTION It is irritating to the eyes,
23
βOAc NHAc Eq OAc
Single 8.5 C-4
67
78
skin and respiratory system; wear suitable
protective clothing and rinse immediately if
30
H
H
Eq OAc
Double 8.5 C-4
68
94
contact occurs.
• Lipase from Aspergillus niger (ANL; Sigma35
H
H
Ax OAc
Double 8.5 C-4
72
80
Aldrich, cat. no. 62301)
• Lecitase ultra (LECI; Novozymes, cat. code
Ax, axial; DP, deprotected position; Eq, equatorial.
a
6-OH alcohol solution was incubated at 4 °C. See Supplementary Table 4 for the full analytical data.
Lecitase Novo)
1786 | VOL.7 NO.10 | 2012 | nature protocols
protocol
© 2012 Nature America, Inc. All rights reserved.
Table 5 | Synthesis of 3-hydroxy-tetraacetylated monosaccharides by acyl-chemical
migration from the 6-OH monodeprotected tetraacetylated products.
pNPB (50 mM) stock solution Dissolve
104.6 mg of pNPB in pure acetonitrile to a final
volume of 10 ml. The solution can be stored for
up to 2 months at − 20 °C.
Substrate
R1
R2
R3
X
pHa DP
Product
Yield (%)
Hydrophobic adsorption buffer Hydrophobic
adsorption buffer is 5–25 mM sodium phos20
αOAc
OAc
Eq OAc Single 9.5 C-3
73
30
phate (pH 7). Dissolve 0.344–1.72 g of sodium
dihydrogen phosphate 1-hydrate in distilled water
31
αOAc
OAc
Ax OAc Single 9.5 C-3
77
50
to a final volume of 0.5 liter and set the pH to
7 by adding, dropwise, a solution of 1 N NaOH
32
βOAc
OAc
Ax OAc Single 9.5 C-3
78
50
in water. The buffer can be stored for 1 week at
Ax, axial; DP, deprotected position; Eq, equatorial.
a
room temperature (~25 °C).
6-OH alcohol solution was incubated at 4 °C. See Supplementary Table 5 for the full analytical data.
Covalent attachment incubation buffer Dissolve
1.72 g of sodium dihydrogen phosphate 1-hydrate
in distilled water to a final volume of 0.5 liter and set the pH to 7 by adding,
• n-Hexane (Merck, cat. no. 104367) ! CAUTION It is highly flammable and
irritating to skin. It is toxic and harmful; there is a danger of serious damage
dropwise, a solution of 1 N NaOH in water. To this solution, add 0.3 g of
to health by prolonged exposure through inhalation.
CTAB (0.06%, wt/vol; for TLL immobilization) or 0.25 ml of Triton X-100
• NaOH (Panreac, cat. no. 141687)
(0.06%, vol/vol; for the rest of the enzymes) and keep stirring until the
• HPLC-grade acetonitrile (Burdick & Jackson, cat. no. AH015-4)
detergent disappears completely. The buffer can be stored at room tempera• NaCl (USB, cat. no. 21618)
ture for 1 week.  CRITICAL The addition of detergent to the immobilization
• NaN3 (Sigma-Aldrich, cat. no. S2002)
buffer is crucial to avoid the formation of bimolecular aggregates of lipases in
• Toluene (Sigma-Aldrich, cat. no. 244511)
order to obtain an efficient and correct enzyme immobilization yield48.
• Ethanol absolute (Sigma-Aldrich, cat. no. 459844) ! CAUTION It is highly
Enzyme activity buffer Enzyme activity buffer is 25 mM sodium phosphate
flammable; use eye shields, face shields, full-face respirator, gloves and a
(pH 7). Dissolve 3.44 g of sodium dihydrogen phosphate 1-hydrate in
multipurpose combination respirator cartridge respirator filter.
distilled water to a final volume of 1 liter and set the pH to 7 by adding,
• H2SO4 (Sigma-Aldrich, cat. no. 320501) ! CAUTION H2SO4 is corrosive to
dropwise, a solution of 1 N NaOH in water. The buffer can be stored for
metals; it causes skin corrosion and serious eye damage. Use eye shields, face
1 week at room temperature.
shields, full-face respirator, gloves and a multipurpose combination respiraEQUIPMENT SETUP
tor cartridge respirator filter.
Stirring systems The use of suspensions requires the use of mild stirring
• Diethyl ether (Sigma-Aldrich, cat. no. 346136) ! CAUTION It is highly flamsystems necessary in order to have a homogenous suspension when taking
mable and may form explosive peroxides. Repeated exposure may cause skin
samples. These may include, for example, magnetic stirring, mechanical
dryness or cracking.
stirring with a helix, orbital stirring, shakers or Coulter stirrers. When
• HCl
using heterogeneous suspensions, an adequate stirring system must be used
• Deuterated chloroform
in most of the steps (immobilization of the enzymes or enzyme activity
EQUIPMENT
determination); however, this does not need to be used when the enzyme
• Teflon-coated magnetic stir bars (Sigma-Aldrich)
• Sintered glass filter (Pobel)
has been already immobilized.
• Vacuum filtering systems (to recover the supports and immobilized
Packing a silica gel column See Box 1 for details on packing a silica gel
enzymes, these systems are simpler than centrifuges; Pobel)
column (i.e., 4 cm internal diameter (i.d.) × 55 cm length).
• Round-bottom flask (Schott-Duran)
Silica gel TLC analysis See Box 2 for silica gel TLC analysis.
• Vacuum pump (Edwards)
HPLC analysis Generally, the regioselective hydrolysis and the chemical acyl
• Funnel (Schott-Duran)
migration reactions were followed by HPLC using a HPLC spectrum P100
• Pyrex chromatography column (Pobel)
(Thermo Separation products). The column was a Kromasil-C18 (250 ×
• Spectrophotometer UVmini 1240 (Shimadzu)
4.6 mm and 5 µm diameter) from Analisis Vinicos (Spain). Analyses were run
• Rotary evaporator (Büchi)
at 25 °C using an L-7300 column oven and UV detector L-7400 at 210 nm.
• TLC silica gel 60 F254 (EMD, cat. no. 5554-7)
The mobile phase was an isocratic mixture of acetonitrile and sodium
• NMR tube (Norell)
phosphate buffer solution in the specific proportion reported for each
• NMR spectrometer (Bruker, cat. no. AV-400 or AV-500)
product in the Analytical Data section. The flow rate was 1.0 ml min − 1.
• Orbital shaker (Certomat BS-1; Braun Biotech)
• Micropipettes for taking samples
• Separatory funnel (Schott-Duran)
• Analytical reversed-phase column (Kromasil RP18, 250 mm × 4.6 mm,
5 µm diameter; Analisis Vinicos)
• HPLC system with a UV detector (Thermo Finnigan)
• pH STAT DL50 Graphix (Mettler Toledo)
• Mechanical stirrer (IKA)
• Coulter stirrer (J.P. SELECTA)
• Magnetic stirrer (VELP Scientifica)
• pH meter (Teknokroma)
• Becker glass, 200 ml
• Equipment for 1H-NMR and 2D correlation spectroscopy (COSY)
REAGENT SETUP
Enzyme solution CAL-B, TLL, LECI and RML crude extracts are in liquid
form, whereas CRL, PFL and ANL are in the form of a lyophilized powder.
The commercial enzyme crude extracts can be stored for up to 6 months
at 4 °C. Each crude extract has been titrated in its protein concentration by
Bradford’s assay47, yielding the following results: CAL-B: 5 mg of protein per
ml, TLL: 14 mg of protein per ml, LECI: 14 mg of protein per ml, RML: 7 mg
of protein per ml, CRL: 34 mg of protein per gram, PFL: 31 mg of protein per
gram, ANL: 42 mg of protein per gram.
OAc
OH
AcO
AcO
O
One step
OAc
AcHN
AcO
OAc
O
AcO
HO
OAc
OAc
O
AcO
One step
OAc
HO
AcO
AcHN
O
+
HO
AcO
66
OAc
OAc
O
Three steps
OAc
AcHN
66
AcO
OAc
O
Three steps
OAc
AcO
OAc
O
AcO
OAc
OAc
O
AcO
81
OAc
O
OAc
O
AcO
OAc
77
HO
AcO
OAc
O
82
AcO
OAc
O
OAc
O
58
OAc
NHAc
OAc
80 OAc
41
OAc
OAc
O
O
AcHN
O
OAc
O
AcO
OAc
OAc
O
AcO
83
O
O
NHAc
Figure 5 | Chemoenzymatic synthesis of different glycoderivatives starting
from the new monodeprotected building blocks.
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Box
1 | Packing a silica gel column
© 2012 Nature America, Inc. All rights reserved.
1. Mix 110 g of silica gel with an adequate amount of the mobile phase required by the purification of the desired product
(see Analytical Data). The aim is to fill the column with an equilibrated silica plug of 17 cm.
2. Swirl the mixture to make a suspension of the silica gel in the solvent.
3. Pour the well-mixed mixture into a column (4 cm i.d. × 55 cm length) in a single smooth action.
4. Pack the silica gel under pressure.
5. Dissolve the crude mixture with the minimum amount of the same mobile phase used to pack the column and load it on top
of the silica bed.
 CRITICAL STEP A thin and compact column front is necessary for good separation of the reaction products.
6. Elute the product and collect fractions of 20–25 ml.
7. Identify the fractions containing the desired product by silica gel TLC using the same mobile phase used during the purification.
Products are visualized by spraying the TLC with a solution of H2SO4 5% (vol/vol) in ethanol and subsequent heating on a hot plate.
8. Collect the desired fractions and evaporate the solvent using a rotary evaporator.
9. Dry the residue under reduced pressure to obtain the purified product as white solids in the yields listed in Table 1 and
Supplementary Table 1. The products can be further recrystallized from cold diethyl ether.
Box
2 | Silica gel TLC analysis
1. Carefully cut a 4 cm × 10 cm silica-gel 60 F254 aluminum-backed TLC plate.
2. Spot a small amount of the sample on the plate.
3. Develop the plate using the eluent reported for each product in the Analytical Data section.
4. Detect the products by completely spraying the plate with a 5% (vol/vol) solution of H2SO4 in ethanol.
5. Let it dry for 2–3 min.
6. Subsequently, heat the TLC on a hot plate and some new light brown spots will appear on the plate surface.
! CAUTION The developing solvents are irritants and should be handled in a fume hood while wearing safety goggles.
PROCEDURE
Peracetylation of carbohydrates ● TIMING 2–5 h
1| Weigh 20 mmol (1.0 equiv.) of the desired unprotected carbohydrate (1, 3, 5–10, 12, 14, 15, 17 and 18; Table 1 and
Supplementary Table 1) into a 250-ml round-bottom flask containing a Teflon-coated magnetic stir bar.
2| Transfer 50 ml of freshly dried pyridine (from commercial anhydrous pyridine stored in a sealed bottle over 3-Å molecular sieves and purged with N2) into the flask with a glass graduated cylinder.
3| Add 1 liter equiv. of Ac2O for each free hydroxyl group of the substrate added with a glass graduated cylinder and turn
the magnetic stirrer on.
4| To the suspension, add a catalytic amount of DMAP (about 0.1 mmol for 20 mmol of unprotected carbohydrate).
 CRITICAL STEP The reaction can be proportionally scaled up or down on the basis of the moles of the limiting substrates
(unprotected carbohydrate), while keeping the proportion of each component constant (Steps 1–3).
5| Cap the flask and keep stirring at room temperature for 3–6 h.
 CRITICAL STEP Normally, the peracetylation reaction is complete once the starting suspension turns to a homogenous
clear solution.
 PAUSE POINT The reaction mixture can be left at room temperature overnight.
6| Verify the complete consumption of the starting material by silica TLC using the mobile phase described for each product in Table 1 and Supplementary Table 1. To visualize by TLC, spray the material with a solution of 5% H2SO4 (vol/vol) in
ethanol and subsequently heat it on a hot plate until the spots appear.
7| After complete consumption of the substrate (as monitored by TLC), add an equal volume of toluene (50 ml) to the flask
(resulting in an azeotropic mixture) and stir for 10 min.
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8| Remove the Teflon-coated magnetic stir bar and evaporate the azeotropic mixture under reduced pressure at 40 °C.
9| Repeat Steps 7 and 8 until pyridine disappears completely.
 CRITICAL STEP Ensure that pyridine is removed completely.
© 2012 Nature America, Inc. All rights reserved.
10| The glassy oil obtained can be further recrystallized from 95% cold ethanol or cold diethyl ether to obtain the
peracetylated compounds (20, 22, 24–29, 31, 33, 34, 36 and 37) as white solids in excellent yields ( > 95%; Table 1 and
Supplementary Table 1).
? TROUBLESHOOTING
11| Characterize the peracetylated products by 1H-NMR and 2D-COSY spectroscopy using deuterated chloroform as
the solvent.
 CRITICAL STEP The α-configuration of the anomeric acetyl ester can be verified by 1H-NMR by searching for a well-defined
doublet between 6 and 7 p.p.m. with an approximated J constant value of 4 Hz. In contrast, the β-configuration is characterized by a doublet placed at 5.38–5.80 p.p.m. with a J constant value of about 8 Hz. For example, in the case of 1,2,3,4,
6-penta-O-acetyl-α-d-glucopyranose (20), the anomeric signal is characterized by a doublet placed at 6.32 p.p.m. with a
J value of 4 Hz, whereas in the case of 1,2,3,4,6-penta-O-acetyl-β-d-glucopyranose (21) the anomeric signal is characterized
by a doublet placed at 5.75 p.p.m. with a J value of 8.1 Hz.
 PAUSE POINT The peracetylated compounds can be stored in a freezer at − 20 °C for 6 months.
Activity assay of lipases ● TIMING 10 min
12| Withdraw an aliquot from the pNPB 50 mM stock solution in acetonitrile prepared as previously described and let it cool
down to room temperature.
13| Add 2.5 ml of enzyme activity buffer to a spectrophotometric cell containing a round Teflon-coated stirrer, start stirring
and preincubate the mixture at 25 °C for 2 min.
14| Add 20 µl of pNPB stock solution to the spectrophotometric cell and homogenize the solution by stirring it for
about 30 s.
15| Add 0.05 ml of lipase solution or suspension to the enzymatic assay solution.
16| Measure esterasic lipase activity using an ultraviolet spectrophotometer by measuring the increase in absorbance (Abs)
at 348 nm (the isosbestic point of p-nitrophenol) produced by the release of p-nitrophenol (extinction molar coefficient:
∈ = 5.150 M − 1 cm − 1) in the hydrolysis of pNPB during the time.
17| Calculate the activity (A) by using the following equation:
A = ∆Abs / min.VT.∈ − 1.Vs − 1, where VT = volume in milliliters of the assay and Vs = volume of enzyme solution in milliliters.
Enzymatic activity is given as one micromole of p-nitrophenol released per minute per milligram of enzyme (IU) under the
conditions described above.
Preparation of lipase-immobilized biocatalyst via enzyme adsorption on octyl-Sepharose support ● TIMING 4 h
18| Wash 1 g of commercial octyl-Sepharose support (Fig. 2) with three volumes of distilled water using a sintered
glass filter.
19| Equilibrate the support with one volume of hydrophobic adsorption buffer (see Reagent Setup).
20| Dry the support for 1 min on the filter.
21| Prepare a lipase solution by mixing an adequate amount of crude lipase (solid or liquid, depending from the physical
state of the commercial catalyst; see Reagent Setup) in 20 ml of hydrophobic adsorption buffer, obtaining a final lipase
concentration of 0.5 mg ml − 1.
22| Add 1 g of the hydrophobic support obtained in Step 20 to the 20 ml of the enzymatic solution prepared in Step 21.
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23| Gently stir the mixture for 3 h at 25 °C under mechanical stirring.
 CRITICAL STEP Magnetic stirring must be avoided in order to prevent the grinding effect of the Teflon stir bar on the
heterogeneous support. Mechanical stirring is recommended.
24| Periodically take samples of the supernatant and suspension and analyze these by the enzymatic activity assay described
in Steps 12–17. The comparison between supernatant and suspension activities will determine the lipase adsorption course.
Once the lipase activity totally disappears from the supernatant, the biocatalyst adsorption can be considered complete.
 CRITICAL STEP To take samples of immobilized enzymes, it is necessary to use cut tips in the pipettes and to stir
vigorously to ensure full dispersion.
© 2012 Nature America, Inc. All rights reserved.
25| Filter the suspension by vacuum on a sintered glass filter and wash the solid at least five times with 50 ml of
distilled water.
26| Remove as much water as possible from the lipase derivative and store it at 4 °C.
 PAUSE POINT The lipase derivatives can be stored for 1 week in the refrigerator without loss of activity. They can also
be stored for a longer time. In this case, with the aim of preventing any microbial contamination, it is recommended to
wash it once with 50 ml of a 0.05% (vol/vol) NaN3 water solution on a sintered glass filter and dry it before storing it in the
refrigerator. In this case, the enzyme derivatives can be stored for up to 1 month without loss of activity. It is important to
wash these derivatives with abundant distilled water before their use in any reaction.
Preparation of lipase-immobilized biocatalyst via enzyme covalent attachment on CNBr-activated Sepharose
● TIMING 4–7 h
27| Activation of the commercial support (Steps 27–29). Suspend 0.4 g of the commercial CNBr-activated Sepharose
lyophilized powder in 20 ml of distilled water and adjust the pH to between 2 and 3 by adding 1 N HCl solution to activate
and to swell the support.
 CRITICAL STEP The commercial lyophilized powder normally swells 2.5–3 times with respect to the dry weight.
Consequently, to use 1 g of swelled-activated support, it will be necessary to start from at least 0.4 g of dry powder.
 CRITICAL STEP The reaction can be proportionally scaled up, while keeping the proportion of each component constant
(Steps 27, 30 and 34).
28| Leave the suspension on mechanical stirring at ambient temperature for a minimum of 1 h.
 PAUSE POINT The activation of the CNBr-Sepharose can be prolonged up to 5 h without any inactivation of the support.
29| Filter the suspension by vacuum on a sintered glass filter.
 CRITICAL STEP It is crucial to avoid washing the recovered activated support with water. This is because the reactive
imido carbonate groups formed in strong acidic medium could be hydrolyzed45.
 PAUSE POINT The activated support can be stored at 4 °C for up to 5 h before its use, after which it starts to degrade.
30| Add 1 g of the swelled CNBr-activated Sepharose support (Steps 27–29) to 20 ml of lipase dissolved in the covalent
incubation buffer under the same conditions reported in Step 21, and set the pH value to 7.
 CRITICAL STEP The immobilization can be proportionally scaled up, while maintaining the proportion of each component
constant.
31| Stir gently for 3 h at 25 °C under mechanical stirring.
 CRITICAL STEP Magnetic stirring must be avoided in order to prevent the grinding effect of the Teflon stir bar on the
heterogeneous support. Mechanical stirring is recommended. Alternatively, a Coulter stirrer can be used.
32| Periodically take samples of the supernatant and suspension and analyze these using the enzymatic activity assay
described in Steps 12–17. Compare the activities in the supernatant and suspension to follow the course of the lipase
adsorption. Once the lipase activity totally disappears from the supernatant, the biocatalyst immobilization can be
considered complete.
 CRITICAL STEP To take samples of immobilized enzymes, it is necessary to use pipette tips that have been cut and to stir
vigorously to ensure full dispersion.
33| Filter the suspension by vacuum on a sintered glass filter, avoiding washing with distilled water.
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34| Resuspend the lipase derivative in 20 ml of 1 M ethanolamine (pH 8) solution to block the residual active groups of
the support.
 CRITICAL STEP The immobilization can be proportionally scaled up, while maintaining the proportion of each
component constant.
35| Gently stir for 90 min at 25 °C under mechanical stirring.
36| Filter the suspension by vacuum on a sintered glass filter and wash the covalent lipase derivative with abundant
distilled water.
© 2012 Nature America, Inc. All rights reserved.
37| Remove as much water as possible from the lipase derivative and store it at 4 °C.
 PAUSE POINT The lipase derivatives can be stored for a few days in the refrigerator without loss of activity. They can also
be stored for a longer time. In this case, with the aim of preventing any microbial contamination, it is recommended to wash
it once with 50 ml of a 0.05% NaN3 water solution on a sintered glass filter before storing it in the fridge. In this case, the
enzyme derivatives can be stored for up to 1 month without loss of activity. It is important to remove the preservative before
using the lipase derivatives in any catalyzed reaction by washing these with abundant distilled water.
Preparation of 6-OH monodeprotected acetylated carbohydrates ● TIMING 10–72 h
38| Dissolve 2.5 mmol of the desired peracetylated substrate (20–28, 30–35, 37–38) to make a 100-ml solution in 50 mM
phosphate buffer with 20% acetonitrile at pH 5.
39| Add 5 g of the selected biocatalyst (Table 2 and Supplementary Table 2) to initialize the reaction.
40| Maintain the hydrolysis reaction under mechanical stirring at 25 °C and keep the pH value constant by automatic
titration using a pH STAT DL50 Graphix with a 6 N NaOH solution in order to avoid the chemical acyl migration.
41| To check the reaction course, monitor it by TLC (generally, Rf ≈ 0.25 of 6-OH monodeprotected carbohydrates) or HPLC
(Supplementary Figs. 1–3) by following the general procedure described in Supplementary Table 2.
42| Once the maximum yield of the monodeprotected product has been reached, filter the reaction mixture by vacuum on a
sintered glass filter.
? TROUBLESHOOTING
43| Saturate the recovered solution with NaCl (about 30–35 g).
44| Transfer the filtrate into a 500-ml separatory funnel.
45| Extract the aqueous layer with ethyl acetate (3 × 100 ml).
46| Combine the organic layer, wash it with saturated aqueous sodium hydrogen carbonate (2 × 100 ml) and with brine
(1 × 100 ml) and dry it by adding approximately 1.5 g of anhydrous sodium sulfate; thereafter, shake mildly for 30 s, filter
the mixture under gravity through a fluted filter paper on a funnel to remove the sodium sulfate and collect the filtrate in a
500-ml round-bottomed flask.
47| Evaporate the solvent using a rotary evaporator at 40 °C under aspirator vacuum (200 mbar) to give the monodeprotected alcohols (39–55), generally as white solids, in excellent yields (Table 2 and Supplementary Table 2). If the residue
obtained is a glassy syrup, it can be further crystallized with cold diethyl ether.
 CRITICAL STEP Normally, the regioselective enzymatic hydrolysis permits one to achieve pure monodeprotected products
that can be used without any further purification. In cases in which the products obtained are impure (40, 42, 44–47, 51,
54–55), it is necessary to perform purification by flash chromatography by following the general procedure described in
Supplementary Table 2.
 PAUSE POINT The round-bottom flask can be sealed with Parafilm or a rubber stopper and stored in a 4 °C refrigerator
overnight or for several days before purification by silica gel.
48| Characterize the products obtained in Step 47 by 1H-NMR and 2D-COSY spectroscopy using deuterated chloroform as solvent.
 PAUSE POINT The pure 6-hydroxy product can be stored at − 20 °C for 2–3 months.
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Preparation of 1-OH monodeprotected acetylated carbohydrates ● TIMING 10–72 h
49| Perform Steps 38–47 using substrates 21, 23, 32 or 36 and the proper corresponding biocatalyst to initialize the
reaction (Table 3 and Supplementary Table 3).
50| Purify the 1-OH-monodeprotected carbohydrates by flash chromatography by following the general procedure described
in Supplementary Table 3 to obtain the monodeprotected alcohols (60–63), generally as white solids, in excellent yields
(Table 3 and Supplementary Table 3).
51| Characterize the products obtained by 1H-NMR and 2D-COSY spectroscopy using deuterated chloroform as solvent.
 PAUSE POINT The pure 1-hydroxy product can be stored at − 20 °C for 2–3 months.
Preparation of 4-OH monodeprotected acetylated product 56 ● TIMING 48 h
52| Perform Steps 38–48 using ANL adsorbed on octyl-Sepharose as the biocatalyst in the hydrolysis of 29. In this way, the
product 56, bearing a free hydroxyl group at C-4, was obtained in 96% yield (Table 3 and Supplementary Table 3).
© 2012 Nature America, Inc. All rights reserved.
Preparation of 4-OH monodeprotected acetylated carbohydrates ● TIMING 3–11 h
53| By using substrates 20–23, 30–33 and 35, perform Steps 38–42 (Table 2 and Supplementary Table 2).
54| Put each of the re-collected solutions of the different products (39–42, 48–51, 53) in a separate 200-ml Becker glass
and allow them to cool down to 4 °C with ice.
55| Put a Teflon-coated magnetic stir bar in each Becker glass and switch the magnetic stirring on.
56| Increase the pH value up to 8.5 by adding a 6 N NaOH solution while maintaining the solution on ice.
57| To check the reaction course, monitor it by HPLC (Supplementary Figs. 1 and 2) following the general procedure
described in Table 4 and Supplementary Table 4.
58| Once the maximum conversion to the 4-OH monodeprotected product has been reached, lower the pH of the solution
down to 5 with 1 N HCl solution and allow it to warm up to room temperature.
59| Saturate the solution with NaCl, transfer the filtrate into a 500-ml separatory funnel and extract the aqueous layer with
ethyl acetate (3 × 100 ml).
60| Combine the organic layer, wash it with saturated aqueous sodium hydrogen carbonate (2 × 100 ml) and with brine
(1 × 100 ml) and dry it by adding approximately 1.5 g of anhydrous sodium sulfate; shake mildly for 5 min, filter the mixture
under gravity through a fluted filter paper on a funnel to remove the sodium sulfate and collect the filtrate in a 500-ml
round-bottomed flask.
61| Evaporate the solvent using a rotary evaporator at 40 °C under aspirator vacuum (200 mbar) to obtain the
monodeprotected alcohol mixture of 3-OH- and 4-OH-alcohols, generally as a glassy syrup.
62| Separate the regioisomer mixture by flash chromatography by following the general procedure described in
Supplementary Table 4 (generally, Rf ≈ 0.32 for 4-OH monodeprotected carbohydrates and Rf ≈ 0.18 for 3-OH
monodeprotected carbohydrates, Supplementary Fig. 3). Thus, the monodeprotected 4-OH alcohols (64–72) were obtained
in excellent yields (Table 4 and Supplementary Table 4).
 PAUSE POINT The round-bottom flask can be sealed with Parafilm or a rubber stopper and stored in a 4 °C refrigerator
overnight or for several days before purification by silica gel.
63| Characterize the products obtained in Step 62 by 1H-NMR and 2D-COSY spectroscopy using deuterated chloroform
as solvent.
 PAUSE POINT The purified 4-OH products can be stored at − 20 °C for 2–3 months.
Preparation of 3-OH monodeprotected acetylated product 59 ● TIMING 72 h
64| In the case of substrate 38, perform Steps 38–48 using RML adsorbed on octyl-Sepharose as biocatalyst. In this way,
the product 59, bearing a free hydroxyl group at C-3 in 95% yield, was achieved (Table 3 and Supplementary Table 3).
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Preparation of 3-OH monodeprotected acetylated carbohydrates ● TIMING 3–11 h
65| By using substrates 20–23, 31–33 and 35, perform Steps 38–42 (Table 2 and Supplementary Table 2).
66| Put each of the re-collected solutions of the different products (39–42, 49–51, 53) in a 200-ml Becker glass and allow
them to cool down to 4 °C with ice.
67| Put a Teflon-coated magnetic stir bar in each Becker glass and switch the magnetic stirring on.
68| Increase the pH value up to 9.5 by adding a 6 N NaOH solution while maintaining them on ice.
 CRITICAL STEP With the aim of obtaining the highest amount of 3-OH alcohol, it is crucial to reach a pH of 9.5 as soon as possible.
For this reason, the use of an automatic titrator (pH STAT DL50 Graphix) with a 6 N NaOH solution is highly recommended.
69| To check the reaction course, monitor it by HPLC (Supplementary Figs. 1 and 2) following the general procedure
described in Table 5 and Supplementary Table 5.
© 2012 Nature America, Inc. All rights reserved.
70| Once the maximum conversion to the 3-OH monodeprotected product has been reached, lower the pH of the solution
down to 5 with 1 N HCl solution and allow the reaction mixture to warm up to room temperature.
71| Perform Steps 59–61.
72| Separate the regioisomer mixture by flash chromatography by following the general procedure described in
Supplementary Table 5 (generally, Rf ≈ 0.32 for 4-OH monodeprotected carbohydrates and Rf ≈ 0.18 for 3-OH
monodeprotected carbohydrates, Supplementary Figs. 2 and 3). Thus, the monodeprotected 3-OH alcohols (73–79) were
obtained (Table 5 and Supplementary Table 5).
 PAUSE POINT The round-bottom flask can be sealed with Parafilm or a rubber stopper and stored in a 4 °C refrigerator
overnight or for several days before purification by silica gel.
73| Characterize the products obtained in Step 72 by 1H-NMR and 2D-COSY spectroscopy using deuterated chloroform
as solvent.
 PAUSE POINT The purified 3-OH products can be stored at − 20 °C for 2–3 months.
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 6.
Table 6 | Troubleshooting table.
Step
Problem
Possible reason
Solution
10
Separation of anomers of
peracetylated lactose
Very similar Rf, separation by flash
chromatography not possible
Separation by preparative TLC
42
Low yield of 50
Enzyme inhibition by the product
Wash the immobilized biocatalyst with abundantly distilled
water or use freshly prepared biocatalyst
● TIMING
Steps 1–11, peracetylation of carbohydrates: 2–5 h
Steps 12–17, activity assay of lipases: 10 min
Steps 18–26, preparation of lipase-immobilized biocatalyst via enzyme adsorption on octyl-Sepharose support: 4 h
Steps 27–37, preparation of lipase-immobilized biocatalyst via enzyme covalent attachment on CNBr-activated Sepharose: 4–7 h
Steps 38–48, preparation of 6-OH monodeprotected acetylated carbohydrates: 10–72 h
Steps 49–51, preparation of 1-OH monodeprotected acetylated carbohydrates: 10–72 h
Step 52, preparation of 56: 48 h
Steps 53–63, preparation of 4-OH monodeprotected acetylated carbohydrates: 3–11 h
Step 64, preparation of 59: 72 h
Steps 65–73, preparation of 3-OH monodeprotected acetylated carbohydrates: 3–11 h
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ANTICIPATED RESULTS
Analytical data
See Supplementary Table 1 for analytical data for compounds 20–38 (corresponding to Steps 1–11). The data for compound
20 are included here as an example, and representative examples with TLC data are shown in Table 1.
1,2,3,4,6-Penta-O-acetyl--D-glucopyranose (20)
H-NMR (500 MHz, CDCl3) δ: 6.32 (d, J = 4.0 Hz, 1H, H-1), 5.45 (t, J = 9.4 Hz, 1H, H-3), 5.15 (t, J = 9.5 Hz, 1H, H-4),
5.10 (dd, J = 9.4, 4.0 Hz, 1H, H-2), 4.10–4.26 (dd, J = 6.0, 3.0, 12.5 Hz, 2H, H-6A, H-6B), 4.10 (m, 1H, H-5), 2.00–2.25
(5s, 15H, CH3).
Analytical data for compound 39 are included below as an example of 6-hydroxy deprotection corresponding to
Steps 38–48 of the procedure. See Supplementary Table 2 for analytical data for compounds 39–55.
1
© 2012 Nature America, Inc. All rights reserved.
12,3,4-Tetra-O-acetyl--D-glucopyranose (39)
TLC hexane:AcOEt 5:5, vol/vol.
HPLC (NH4H2PO4 10 mM buffer:ACN 7:3, vol/vol, pH 4) Rt = 7.7 min.
1
H-NMR (500 MHz, CDCl3) δ: 6.38 (d, J = 3.7 Hz, 1H, H-1), 5.56 (t, J = 9.9 Hz, 1H, H-3), 5.14 (t, J = 9.9 Hz, 1H, H-4),
5.10 (dd, J = 3.7, 9.9 Hz, 1H, H-2), 3.96 (m, 1H, H-5), 3.61–3.75 (dd, J = 4.2, 2.4, 12.9 Hz, 2H, H-6A, H-6B),
2.00–2.25(4s, 12H, CH3).
Analytical data for compound 60 are included below as an example of 1-hydroxy deprotection corresponding to
Steps 49–51 of the procedure. See Supplementary Table 3 for analytical data for compounds 60–63.
23,4,6-Tetra-O-acetyl-/-D-glucopyranose (60)
TLC hexane:AcOEt 5:5, vol/vol.
HPLC (NH4H2PO4 10 mM buffer:ACN 6:4, vol/vol, pH 4) Rt = 8.5 min.
1
H-NMR (500 MHz, CDCl3) δ: 5.47 (t, J = 9.7 Hz, 1H, H-3α), 5.38 (d, J = 3.5 Hz, 1H, H-1α), 5.18 (t, J = 9.6 Hz, 1H,
H-3β), 5.03 (t, J = 9.7 Hz, 2H, H-4α, H-4β), 4.86 (dd, J = 8.1, 9.7 Hz, 1H, H-2β), 4.83 (dd, J = 3.6, 10.1 Hz, 1H, H-2α),
4.70 (d, J = 8.0 Hz, H-1β), 3.95–4.25 (m, 3H, H-5α, 2H-6α/β), 3.71 (m, 1H, H-5β), 1.90–2.20 (s, 12H, 4CH3).
Analytical data for compounds 56 and 64 are included below as representative examples of 4-hydroxy deprotection
corresponding to Steps 52–63 of the procedure. See Supplementary Tables 3 and 4 for analytical data for compounds
56, 64–72.
4-Nitrophenyl-2,3-di-O-acetyl--D-xylopyranoside (56)
TLC hexane:AcOEt 4:6, vol/vol.
HPLC (NH4H2PO4 10 mM buffer:ACN 6:4, vol/vol, pH 4) Rt = 8.3 min.
1
H-NMR (500 MHz, CDCl3) δ: 8.10 (d, J = 8.4 Hz, 2H, H-3′, H-5′), 6.88 (d, J = 8.3 Hz, 2H, H-2′, H-6′), 5.21 (d, J = 5.3
Hz, 1H, H-1), 5.16 (m, 1H, H-2), 4.78 (m, 1H, H-3), 4.12 (m, 1H, H-5), 3.92 (m, 1H, H-4), 3.55 (m, 1H, H-5), 2.10
(s, 3H, CH3), 2.05 (s, 3H, CH3).
12,3,6-Tetra-O-acetyl--D-glucopyranose (64)
TLC hexane:AcOEt 5:5, vol/vol.
HPLC (NH4H2PO4 10 mM buffer:ACN 8:2, vol/vol, pH 4) Rt = 17.5 min.
1
H-NMR (400 MHz, CDCl3) δ: 6.23 (d, J = 3.4 Hz, 1H, H-1), 5.28 (t, J = 9.8 Hz, 1H, H-3), 4.96 (dd, J = 10.2, 3.7 Hz, 1H,
H-2), 4.21–4.41 (m, J = 8.6, 3.9, 12.6 Hz, 2H, H-6A, H-6B), 3.92 (m, 1H, H-5), 3.59 (t, J = 9.3 Hz, 1H, H-4), 1.96–2.11
(s, 12H, 4CH3).
Analytical data for compounds 57 and 73 are included below as representative examples of 4-hydroxy deprotection
corresponding to Steps 64–73 of the procedure. See Supplementary Tables 3 and 5 for analytical data for Compounds
57–59, 73–79.
46-Di-O-acetyl-D-glucal (57)
TLC hexane:AcOEt 5:5, vol/vol.
HPLC (NH4H2PO4 10 mM buffer:ACN 7:3, vol/vol, pH 4) Rt = 5.2 min.
1
H-NMR (500 MHz, CDCl3) δ: 6.42 (dd, J = 6.1 Hz, 1H, H-1), 4.95 (dd, J = 6.2, 2. Hz, 1H, H-4), 4.84 (dd, J = 6.2, 3.2,
2.7 Hz, 1H, H-2), 4.43 (ddd, J = 6.7, 5.3 Hz, 1H, H-5), 4.22–4.38 (m, 2H, H-6A, H-6B), 4.20–4.11 (dd, J = 6.2, 2.2 Hz,
1H, H-3), 2.55 (bs, 1H, OH), 2.16 (s, 3H, CH3), 2.11 (s, 3H, CH3).
1794 | VOL.7 NO.10 | 2012 | nature protocols
protocol
12,4,6-Tetra-O-acetyl--D-glucopyranose (73)
TLC hexane:AcOEt 5:5, vol/vol.
HPLC (NH4H2PO4 10 mM buffer:ACN 8:2, vol/vol, pH 4) Rt 15.5 min.
1
H-NMR (400 MHz, CDCl3) δ: 6.33 (d, J = 3.5 Hz, 1H, H-1), 5.45 (t, J = 9.8 Hz, 1H, H-4), 5.18 (dd, J = 10.2, 3.7 Hz, 1H,
H-2), 4.31 (t, J = 9.3 Hz, 1H, H-3), 4.05–4.27 (dd, J = 4.0, 2.1, 12.6 Hz, 2H, H-6A, H-6B), 4.15 (m, 1H, H-5), 2.00–2.20
(s, 12H, 4CH3).
Note: Supplementary information is available in the online version of the paper.
Acknowledgments This work was supported by The Spanish National Research
Council (CSIC) and the Spanish Ministry of Science. We acknowledge Á. Berenguer
(Instituto Universitario de Materiales, Universidad de Alicante) for his help
during the writing of this paper.
© 2012 Nature America, Inc. All rights reserved.
AUTHOR CONTRIBUTIONS M.F. and J.M.P. performed the experiments; M.F. and
J.M.P. analyzed data; J.M.P. and M.F. wrote the manuscript; J.M.P. and J.M.G.
designed the study and experiments; and M.F. and M.T. designed the study for
glycoderivative synthesis.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Published online at http://www.nature.com/doifinder/10.1038/nprot.2012.098.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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