Molecular sieves mediated green per-O-acetylation of carbohydrate templates and lipase catalyzed regioselective alcoholysis of 2,3,5-Tri-O-acetyl-D-ribonolactone

Article abstract of DOI:10.5935/0103-5053.20150037

The per-O-acetylation of D-ribono-1,4-lactone and representative carbohydrates through the combination of acetic anhydride and molecular sieves under solvent-free conditions is demonstrated. The use of 13X/KCl molecular sieves as the heterogeneous catalyst was found to be more efficient than the excess of pyridine normally employed in the conventional method, giving high yields of the expected peracetylated product after 3 h at 25 °C or 1 h at 50 °C. The transformation can be carried out in gram scale and in an open flask. Additionally, the catalyst is readily separated from the reaction medium and can be reutilized without significant loss of activity. This green procedure for acetylation was extended to D-ribonolactone derivatives and natural carbohydrates. To demonstrate the synthetic utility of the method, 2,3,5-tri-O-acetyl-D-ribonolactone was selected as the substrate for the regioselective alcoholysis of acetyl group catalyzed by Candida antarctica lipase B in EtOH to selectively produce 2,3-di-O-acetyl-D-ribonolactone in gram scale.

Full text of DOI:10.5935/0103-5053.20150037

http://dx.doi.org/10.5935/0103-5053.20150037
J. Braz. Chem. Soc., Vol. 26, No. 4, 755-764, 2015.
Printed in Brazil - ©2015 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
Molecular Sieves Mediated Green Per-O-Acetylation of Carbohydrate Templates and
Lipase Catalyzed Regioselective Alcoholysis of 2,3,5-Tri-O-Acetyl-D-Ribonolactone
Herbert M. Cardozo,a Thaís F. Ribeiro,a Marcus M. Sá,b Damianni Sebrão,b,#
Maria G. Nascimentob and Gustavo P. Silveira*,a
aDepartamento de Química Orgânica, Instituto de Química,Universidade Federal do
Rio Grande do Sul, 91501-970 Porto Alegre-RS, Brazil
bDepartamento de Química, Universidade Federal de Santa Catarina,
88040-900 Florianópolis-SC, Brazil
The per-O-acetylation of D-ribono-1,4-lactone and representative carbohydrates through the
combination of acetic anhydride and molecular sieves under solvent-free conditions is demonstrated.
The use of 13X/KCl molecular sieves as the heterogeneous catalyst was found to be more efficient
than the excess of pyridine normally employed in the conventional method, giving high yields
of the expected peracetylated product after 3 h at 25 °C or 1 h at 50 °C. The transformation can
be carried out in gram scale and in an open flask. Additionally, the catalyst is readily separated
from the reaction medium and can be reutilized without significant loss of activity. This green
procedure for acetylation was extended to D-ribonolactone derivatives and natural carbohydrates.
To demonstrate the synthetic utility of the method, 2,3,5-tri-O-acetyl-D-ribonolactone was selected
as the substrate for the regioselective alcoholysis of acetyl group catalyzed by Candida antarctica
lipase B in EtOH to selectively produce 2,3-di-O-acetyl-D-ribonolactone in gram scale.
Keywords: D-ribonolactone, per-O-acetylation, regioselective alcoholysis, CAL-B, molecular
sieves
Introduction
variety of methods3-5 to produce D-ribono-1,4-lactone (2),
which has been widely used as a versatile chiral building
block6,7 for the synthesis of several natural products8-10
as well as compounds relevant to medicinal chemistry
and/or chemical biology, including nucleotides and
C-ribonucleosides as antivirals.11-13
Carbohydrates are considered as appealing chiral
starting materials for asymmetric synthesis not only
because they possess several functionalities and stereogenic
centers that are amenable to a variety of transformations
but also due to their relatively low cost and availability
from sustainable feedstocks.1 Nevertheless, one of the
main drawbacks associated with the use of natural sugars
as building blocks for the synthesis of more elaborated
compounds is the possible coexistence of different isomers
in solution, including cyclic a- and b-lactols and also
ring-opening structures.2 On the other hand, the oxidation
of the anomeric center in pentoses and hexoses to give
the corresponding lactones is an elegant strategy to solve
the problem of dealing with several isomers in solution.
Therefore, D-ribose (1, Figure 1) can be oxidized by a
O
O
OH
O
HO
HO
HO
OH
HO
OH
2
1
O
O
O
O
O
O
HO
O
O
O
O
O
O
O
O
3
4
Figure 1. Structures of D-ribose (1), D-ribono-1,4-lactone (2), 2,3,5-tri-
O-acetyl-D-ribonolactone (3) and 2,3-di-O-acetyl-D-ribonolactone (4).
Another drawback related to the widespread use of
carbohydrates as synthetic building blocks is the presence
of hydroxyl groups of similar reactivity, which frequently
requires the application of protocols for hydroxyl protection
*e-mail: gustavo.silveira@iq.ufrgs.br
#Current address: Centro de Ciências Tecnológicas da Terra e do Mar,
Universidade do Vale do Itajaí (CCTMar-UNIVALI), 88302-901 Itajaí-
SC, Brazil

756
Molecular Sieves Mediated Green Per-O-Acetylation of Carbohydrate Templates
J. Braz. Chem. Soc.
and deprotection.14 Per-O-acetylation of carbohydrates is a
very usual approach to this end.15-17 In fact, fully acetylated
sugars are versatile intermediates for the synthesis of
many naturally occurring glycosides, oligosaccharides
and glycoconjugates. Pyridine is largely used as catalyst
in acylation reactions. However, long-term exposure to
this reagent can cause sterility in males.18 Triethylamine in
combination with 4-(dimethylamino)pyridine (DMAP) and
acetonitrile (ACN) is an important alternative for pyridine
in the per-O-acetylation.19 Nevertheless, the required excess
of reagents and solvents creates further environmental and
economic concerns and also demands aqueous work-up.
Thus, new synthetic methodologies must be developed to
decrease the use of pyridine and its derivatives in organic
synthesis.
Heterogeneous catalysis is being adopted as an
alternative methodology for acylation due to major
advantages related to its use, including the simple
separation of the heterogeneous catalyst from the reaction
flask by filtration at the end of the process. Also, it can
be employed in a new batch without any or minimal
purification/reactivation steps, which obviously represents
economic and environmental advances. Therefore,
molecular sieves (MS), which have several catalytic sites
inside their structures, have been successfully employed
to promote acylation of carbohydrates,15 nucleosides,20
alcohols and phenols.21,22 The basic character of some MS is
usually associated with the existence of negatively charged
oxygen atoms covalently bonded to the Si-Al lattice.23
Lipase B from Candida antarctica (CAL-B) is a widely
used lipase in biocatalyzed transformations exhibiting a
high degree of substrate selectivity and also having the
potential for use in hydrolysis and alcoholysis reactions24
involving acylated derivatives of carbohydrates25-28
and nucleosides.29,30 Similar to MS, this heterogeneous
biocatalyst can be readily separated from the reaction
medium and also reutilized for many cycles.
Results and Discussion
Peracetylation of 2 and representative carbohydrates
Initially, 2,3,5-tri-O-acetyl-D-ribonolactone (3) was
prepared under conventional conditions33 in 65% yield
by treating D-ribonolactone 2 with acetic anhydride using
pyridine as the catalyst at 25 °C for 3 h (Table 1, entry 1).
In addition to the toxicity related to pyridine,18 it is also
important to mention that preparation of 3 by this method (or
using DMAP instead of pyridine)33 requires aqueous work-up
which not only generated large volumes of residues but also
led to considerable mass losses34 of the product with negative
impact in the chemical yield of the process.
To find a suitable heterogeneous catalyst for the
acetylation of D-ribonolactone 2 with acetic anhydride, the
reaction was screened in the presence of four commercial
and two modified MS, which were dried under vacuum for
24 h at 100 °C prior to use. After 2 h, the reactions using
3A, 4A, and 5A MS presented many new spots on the thin
layer chromatography (TLC) plate (Table 1, entries 2-5).
The less polar spot could be ascribed to the per-O-acetylated
product 3, while the more polar spots might be related to the
mono- and/or diacetylated products. Attempts to separate
these partially acylated D-ribono derivatives by column
chromatography did not result in pure products, possibly
due to fast 1,2-acyl migration in solution.35,36 On the other
hand, the use of 13X MS or the potassium-exchanged
13X (hereby denominated as 13X/KCl) led to promising
results after 2 h (Table 1, entries 6 and 7, respectively).
Overwhelmingly, running the reaction for an additional
hour (3 h in entries 8 and 9 instead of 2 h in entries 2-7)
led to an improved formation of the peracetylated lactone 3
when 13X MS was employed as the catalyst (entry 8),
while the use of 13X/KCl under the same conditions was
even more effective and gave 3 in quantitative yield and
high purity (entry 9).
As part of our interest in developing synthetic
methodologies to bypass protocols of protection and
deprotection of carbohydrate derivatives,14 allied with
the development of chemical transformations that do not
require harmful reagents or complex purification techniques
and thus avoid the generation of toxic waste,20,21,23,31,32
we present the solvent-free and pyridine-free per-O-
acetylation of D-ribonolactone 2 (Figure 1) and a set
of representative pentoses, as well as hexoses, through
the reaction with acetic anhydride mediated by MS. In
addition, we developed the regioselective alcoholysis of
2,3,5-tri-O-acetyl-D-ribonolactone (3) catalyzed by CAL-B
to selectively produce 2,3-di-O-acetyl-D-ribonolactone (4,
Figure 1).
The acetylation was also performed by heating the
reaction at 50 °C, with the starting material 2 being
consumed in 1 h (Table 1, entry 10). However, the final yield
of 80% was not as high as that obtained by conducting the
reaction at 25 °C (entry 9), possibly due to the sensitivity of
the D-ribonolactone 2 (as well as their acetylated products)
to elevated temperatures, which might result in some in situ
polymerization (caramelizing reaction) that was eventually
separated in the work-up process.As expected, the reaction
carried out in the absence of any catalyst led to negligible
conversions to the acetylated product (entry 11).
Thus, the present methodology for acetylation using
13X/KCl as the catalyst can be considered as an excellent
replacement for the conventional conditions using toxic

Vol. 26, No. 4, 2015
Cardozo et al.
757
pyridine. Noteworthy, the benefits are not only in terms of
environmental and economic issues, but also in the relative
reaction rates (Table 1, entry 1 versus 8 and 9).
acetylation to some derivatives of D-ribonolactone.
Thus, 2,3-O-isopropylidene-D-ribonolactone3,4,7 (5) and
3,4-O-benzylidene-D-ribonolactone7 (6, Zinner’s lactone)
were readily synthesized by the treatment of 2 with acetone
(25 °C, 15 min) or benzaldehyde (90 °C, 1.5 h) in the
presence of concentrated HCl (Scheme 1).
Table 1. Screening a variety of MS as basic catalysts for the per-O-
acetylation reaction of D-ribonolactone 2 with acetic anhydridea
Proceeding with the acetylation of 5 and 6 with acetic
anhydride using pyridine as the catalyst, at 25 °C, gave
the corresponding products 7 and 8 in yields of 75% and
70%, respectively (Table 2, entries 1 and 4). Switching
pyridine by 13X/KCl and applying the previously
established reaction conditions furnished 7 and 8 in yields
of 60% and 38%, respectively (Table 2, entries 2 and 5).
The low yield observed for the acetylation of the Zinner’s
lactone 6 at 25 °C might be related to its low solubility in
most common solvents at 25 °C. In fact, increasing the
temperature improved considerably the reaction yields in
both cases, with the monoacetylation being quantitatively
achieved after only 1 h to give derivatives 7 and 8 (Table 2,
entries 3 and 6).
O
Ac2O
O
O
O
O
Catalyst
HO
O
O
O
HO
OH
O
O
2
3
entry
Catalyst
Pyridine
3A
Temperature / °C
time / h
Yield / %
1
25
25
25
25
25
25
25
25
25
50
25
3
2
2
2
2
2
2
3
3
1
2
65
b
2
b
b
b
3
4A
4
4A 1000
5A
5
6
13X
61
65
80
99
80
-
7
13X/KCl
13X
8
Acetylation of natural carbohydrates
9
13X/KCl
13X/KCl
None
10
11
After the success observed for the acetylation
of D-ribonolactone 2 and its derivatives 5 and 6, the
methodology was applied to the acetylation of a set of
natural carbohydrates (Table 3). Preliminary TLC studies
showed that the peracetylation of these representative
carbohydrates demand longer periods to complete.
Therefore, treatment of three hexoses (D-glucose,
D-galactose and D-mannose) and two pentoses (D-ribose
and D-xylose) with an excess (20 equiv.) of acetic anhydride
in the presence of pyridine at 25 °C for 24 h furnished the
corresponding per-O-acetylated products as a mixture of a-
and b-anomers in 58-66% yield (Table 3, entries 1, 4, 7, 10,
and 13). Surprisingly, the per-O-acetylated carbohydrates
were obtained in lower yields when pyridine was replaced
by 13X/KCl, under the same conditions (Table 3, entries
2, 5, 8, 11, and 14).
aReaction was followed by TLC using 1:1 EtOAc/hexanes; product 3
stains in vanillin; bthree to four spots were observed in the TLC
plate, which might be related to a mixture of acetylated derivatives of
D-ribonolactone 2.
Because catalyst reutilization is a desirable feature for
industrial processes, the possibility of reusing the 13X/KCl
catalyst was determined by running the peracetylation of
D-ribonolactone 2 with the same catalyst for four cycles.
After each batch, EtOAc was added to the reaction and the
catalyst was separated by filtration. The catalytic activity
of reused 13X/KCl was completely maintained even after
four cycles and the product 3 was consistently obtained in
high yields (89-93%) after every cycle.
Acetylation of D-ribonolactone derivatives
Because all natural carbohydrates under study presented
low solubility in acetic anhydride at 25 °C, this observation
might explain the limited reaction outcome observed when
13X/KCl was used as the catalyst (in comparison with
Inspired by the above results obtained for the 13X/KCl
catalyst, we decided to extend the method of
(CH3)2CO
O
PhCHO
O
O
O
O
O
HCl
HCl
HO
HO
25 o
C
90 o
C
OH
O
HO
OH
1.5 h
15 min
O
O
O
5
2
6
H
Ph
Scheme 1. Synthesis of 2,3-isopropylidenelactone 5 and Zinner’s lactone 6.

758
Molecular Sieves Mediated Green Per-O-Acetylation of Carbohydrate Templates
J. Braz. Chem. Soc.
Table 2. Acetylation of the D-ribonolactone derivatives 5 and 6
O
O
O
O
RO
5 R = H
6 R = H
Ac2O
Catalyst
OR
O
7
8
R = CH3CO
R = CH3CO
O
O
O
H
Ph
entry
Substrate
Catalyst
Pyridine
13X/KCl
13X/KCl
Pyridine
13X/KCl
13X/KCl
Temperature / °C
time / h
Product
Yield / %
75
1
2
3
4
5
6
5
5
5
6
6
6
25
25
50
25
25
80
24
24
1
7
7
7
8
8
8
60
> 99
70
24
24
1
38
> 99
pyridine, which ultimately play the role of a cosolvent).
Therefore, we tested a variety of conditions to improve
the substrate solubility, including the use of ultrasound
bath and the addition of twice the original amount of acetic
anhydride. Unfortunately, no significant improvement in the
reaction rates or yields was noticed after 24 h at 25 °C under
these modified conditions (results not shown). Regarding
the formation of a- and b-diastereoisomers in the product
mixture, with the exception of the per-O-acetylglucose all
D-carbohydrates tested gave similar amounts of a- and
b-anomers when pyridine was used as catalyst (Table 3,
entry 1 versus 4, 7, 10). Conversely, employing 13X/KCl as
the catalyst, at 80 °C, led to the selective (or even exclusive)
formation of the b-anomer, with the exception of the per-
O-acetylmannose, which gave preferentially the a-isomer
(Table 3, entries 3, 6, 12, 15 versus 9). Surprisingly,
the a-anomers of the peracetylated-D-ribose were not
identified by H nuclear magnetic resonance (NMR) at
the end of the reaction using 13X/KCl as the catalyst
(Table 3, entry 15).
Finally, attempts to peracetylate a ketose, namely,
D-fructose, using either pyridine or the present method
with 13X/KCl, at 25 °C, did not result in any consumption
of the starting material by TLC (Table 3, entries 16, 17).
On the other hand, conducting the transformation at high
temperature led to the formation of a complex mixture of
products by 1H NMR (Table 3, entry 18).
the selective hydrolysis of the 5-O-acetyl group from per-
O-acetylated carbohydrates25,26,34,42-44 and nucleosides29,45-47
were reported, in which the best results were obtained in
phosphate buffer. Therefore, experiments were carried
out to evaluate the CAL-B catalyzed hydrolysis of 3 at
physiological conditions (pH 7.0 at 35 °C). While the
TLC supported the formation of a new species after 24 h,
the product was very soluble in the buffer medium and
attempts to extract it with CH2Cl2 or EtOAc failed. As an
alternative work-up, water was previously removed from
the buffer solution by a rotary evaporator, but attempts to
separate out the possible product from the crude residue
with a variety of solvents were all unsuccessful.
The regioselective lipase-catalyzed28,34,45 alcoholysis of
the acetyl group in peracetylated a- and b-D-ribofuranosides
was previously demonstrated; therefore, we moved our
attention to alcoholysis conditions instead of hydrolysis,
and CAL-B was again the enzyme selected to conduct the
experiments. Initially, to determine the minimal amount
of enzyme necessary to catalyze the alcoholysis in a
reasonable time, test reactions starting from 0.5 mmol of
substrate 3 were run in EtOH by adding different quantities
of CAL-B (Table 4).
1
While the conversion rates to the 2,3-di-O-acetyl-D-
ribonolactone (4) were high for reactions using 280 or
140 mg of the commercial enzyme (Table 4, entries 1
and 2), decreasing the amount of CAL-B slows down the
conversion to 4 under the same conditions (Table 4, entries 3,
4, and 5). In all cases, the formation of D-ribonolactone 2
and 3-O-acetyl-D-ribonolactone (9, Scheme 2) were also
noted in the crude NMR due to extensive ethanolysis of
the acetyl group.
Selective deacetylation of 3
As a synthetic application for this mild and green
method to prepare 2,3,5-tri-O-acetyl-D-ribonolactone (3)
and other acetylated carbohydrates, we envisioned that a
chemoenzymatic process should be useful to selectively
produce deacetylated products. Several studies involving
To evaluate the role of the alcoholic solvent, 2,3,5-tri-
O-acetyl-D-ribonolactone (3) was then submitted to
alcoholysis in the presence of CAL-B (35 °C, 150 rpm)

Vol. 26, No. 4, 2015
Cardozo et al.
759
Table 3. Per-O-acetylation of natural carbohydratesa
entry
1
Substrate
Catalyst
Pyridine
13X/KCl
13X/KCl
Pyridine
13X/KCl
13X/KCl
Pyridine
13X/KCl
13X/KCl
Pyridine
13X/KCl
13X/KCl
Pyridine
13X/KCl
13X/KCl
Pyridine
13X/KCl
13X/KCl
Temperature / °C
Yield / %
65
a:b / %b
72:28
ndc
Ref.
37
-
HO
HO
25
25
80
25
25
80
25
25
80
25
25
80
25
25
80
25
25
80
O
OH
2
26
HO
D
OH
-glucose
3
80
21:79
58:42
ndc
-
HO
HO
4
62
38,39
-
O
OH
5
34
HO
OH
6
78
17:83
46:54
ndc
-
D
HO
HO
-galactose
7
66
40
-
O
OH
8
53
HO
OH
-mannose
9
D
79
72:28
57:43
ndc
-
10
11
12
13
14
15
16
17
18
58
41
-
O
OH
HO
35
HO
OH
D
-xylose
84
19:81
ndc
-
64
-
O
OH
HO
20
ndc
-
HO
D
OH
61
0:100d
ndc
33
-
-ribose
-
OH
O
HO
OH
-
ndc
-
HO
OH
ndc
-
D
-fructose
e
aAll reactions were performed for 24 h and followed by TLC using 1:1 EtOAc/hexanes; products stain in vaniline; bH-a and H-b chemical shifts were assigned
based on the literature (references 33, 37-41); cnd = not determined; dproduct obtained as a 57:47 mixture of b-D-ribonopyranose and b-D-ribonofuranose;
the presence of a-anomer was not observed; ecomplex mixture of products was obtained.
using MeOH, EtOH, i-PrOH, and n-BuOH as solvent/
reagent. After the time indicated in Table 5, a clear liquid
aliquot was carefully removed from the reaction flask, the
solvent evaporated to dryness in the rotary evaporator and
the residue analyzed by 1H NMR.
The 1H NMR spectrum of each sample along the time
presents the decrease of the triplet at 4.88 ppm, which
is assigned to the H-4 from the starting peracetylated 3,
and the appearance of a triplet at 4.68 ppm, related to the
H-4 from 2,3-di-O-acetyl-D-ribonolactone (4) formed as
the product. The conversion rate of 2,3,5-tri-O-acetyl-D-
ribonolactone (3) to 2,3-di-O-acetyl-D-ribonolactone (4)
is quite similar in MeOH, i-PrOH, and n-BuOH. For
instance, less than half of the starting material 3 was
consumed after 6 h in i-PrOH (Table 5). The complete
deacetylation of both 3 and 4 in i-PrOH ultimately
furnished D-ribonolactone 2 as the exclusive product
of solvolysis after 18 h (Scheme 2). On the other hand,
the use of EtOH as the solvent/reagent led to fast and
selective reactions, with the conversion of 3 to 4 reaching
75% after only 4 h without the competitive formation of
Table 4. Optimization of the amount of CAL-B for the ethanolysis of
3 in 6 ha
O
CAL-B
O
O
EtOH
O
O
O
O
HO
O
150 rpm
35 oC
6 h
O
O
O
O
O
O
3
4
entry
CAL-B / mg
Conversion to 4 / %
1
2
3
4
5
280
140
70
93
93
81
62
44
35
18
aReaction was run in a 0.5 mmol scale. Conversion was determined by
1H NMR integration (400 MHz, DMSO-d6) of the triplets at 4.88 and
4.68 ppm assigned as the H-4 of 3 and H-4 of 4, respectively.

760
Molecular Sieves Mediated Green Per-O-Acetylation of Carbohydrate Templates
J. Braz. Chem. Soc.
O
CAL-B
O
O
O
O
O
O
O
O
Alcohol
O
O
HO
O
HO
O
HO
+
+
O
O
150 rpm
35 o
O
O
O
O
O
OH
HO
OH
C
3
4
9
2
Scheme 2. CAL-B catalyzed alcoholysis of 2,3,5-tri-O-acetyl-D-ribonolactone (3).
Table 5. Consumption of 3 (%) through solvolysis in an added alcohol
to give 4a
catalyzed ethanolysis (35 °C, 150 rpm, 6 h) cleanly
produced 2,3-di-O-acetyl-D-ribonolactone (4) after
purification by flash chromatography on silica gel using
EtOAc/hexanes 1:3 (570 mg, 48% yield; 14% overall
yield for the three steps).
Alcohol
MeOH
EtOH
0.5 h
16
1 h
19
54
14
19
2 h
25
59
22
20
4 h
33
75
31
32
6 h
37
50
93b
43
O
O
OH
O
3 steps
i-PrOH
n-BuOH
13
HO
HO
O
O
11
52
30%
O
O
HO
OH
aValues obtained by integration of the triplets at 4.88 and 4.68 ppm
4
1
b
assigned as the H-4 of 3 and H-4 of 4, respectively; the competitive
formation of 3-O-acetyl-D-ribonolactone (9) was also observed.
35 o
C
Br2
NaHCO3
H2O
25 oC, 2 h
CAL-B
EtOH
6 h
O
any side products. However, longer reaction time (6 h)
enabled the formation of 3-O-acetyl-D-ribonolactone (9)
in non-negligible amounts, based on the appearance of
triplets at 4.45 and 4.39 ppm corresponding to H-2 and
H-4 in 9 (Scheme 2).
Ac2O
O
O
O
O
13X/KCl
HO
O
O
25 oC, 3 h
O
O
O
HO
OH
2
3
The reusability of immobilized enzymes can determine
the economic viability of a biosynthetic process. Thus,
CAL-B was employed as the catalyst for the alcoholysis
of 3 in five subsequent cycles. At the end of each batch,
CAL-B was removed from the reaction, rinsed with MeOH
andACN and used in the next batch. However, the reaction
rate for the formation of the expected product 4 was not
maintained from cycle to cycle and gradually slowed down
(data not shown). This observation is possibly related to
an extensive inhibition of the lipase activity caused by
prolonged exposure to EtOH, which might be able to
remove the aqueous micro-layer from the enzyme and thus
alter the pH in the catalytic site.48
Scheme 3. Three-step synthesis of 4 from 1.
Conclusions
We have disclosed the use of 13X/KCl as an inexpensive
and green catalyst to substitute toxic pyridine in the per-
O-acetylation of D-ribonolactone 2. Besides promoting
faster acetylation reactions than pyridine without the need
for aqueous work-up, the heterogeneous catalyst can be
efficiently reutilized in subsequent cycles. In addition, the
method was extended to the acetylation of two protected
D-ribonolactone derivatives and also the per-O-acetylation
of several natural carbohydrates, but in the latter cases
it is necessary to warm the reaction to obtain good
yields after 24 h. To further explore the synthetic utility
of the peracetylated carbohydrates, we developed the
regioselective CAL-B catalyzed enzymatic alcoholysis
of 2,3,5-tri-O-acetyl-D-ribonolactone (3) to produce
2,3-di-O-acetyl-D-ribonolactone (4) in good yield under
mild and green conditions (EtOH as the solvent/reagent,
35 °C). Finally, to demonstrate the synthetic utility of the
peracetylation/selective deacetylation procedure, a simple
and efficient three-step route was developed to prepare
gram quantities of 2,3-di-O-acetyl-D-ribonolactone (4)
starting from D-ribose (1), in 14% overall yield and only
one chromatography step, indicating the feasibility of
scaling up the process.
Scaling up: three-step synthesis of 2,3-di-O-acetyl-D-
ribonolactone (4) from D-ribose (1)
After determining the best conditions for the per-
O-acetylation of D-ribonolactone 2 followed by the
regioselective ethanolysis of per-O-acetylated 3 to give
2,3-di-O-acetyl-D-ribonolactone (4), the route was adapted
for a gram scale starting from D-ribose (1, Scheme 3).
Accordingly, 2,3,5-tri-O-acetyl-D-ribonolactone
(3) was prepared by first oxidizing D-ribose (1) to
D-ribonolactone 2 using molecular bromine5,49 followed
by 13X/KCl catalyzed peracetylation reaction with acetic
anhydride (ca. 29% yield, two steps). Subsequent CAL-B

Vol. 26, No. 4, 2015
Cardozo et al.
761
Experimental
with a magnetic stirring bar and then 0.1 mL (2 drops) of
12 mol L-1 HCl was added at 25 °C. After 15 min, 0.5 mL
of CH2Cl2 was poured to dissolve the white solid formed
during the course of the reaction. Then the white mixture
was quenched by pouring 300 mg of powdered NaHCO3
in small portions (5 min, until CO2 effervescence ceases),
which turned the mixture into a yellow color. The liquid
phase was decanted and a second portion of CH2Cl2
(0.5 mL) was added to the reaction residue to extract
additional white solid. This process was repeated a second
time and the combined CH2Cl2 extracts were evaporated to
dryness using a rotary evaporator to give 5 as a colorless
solid (80 mg, 42% yield).
General methods
CAL-B [Novozym 435, 10,000 propyl laurate units
(PLU) mg-1 solid] was donated by NovozymesA/S (Brazil).
The enzyme was used without any further treatment or
purification. D-ribose (1) was acquired from Now™ Sports,
D-ribono-1,4-lactone (2), as well as, 3A, 4A, 5A, and 13X
MS were purchased from Sigma-Aldrich. 13X/KCl was
prepared by the diffusion method using aqueous KCl as
previously reported.20,21 Thermally-modified 4A-1000
catalyst was prepared by heating 4A MS at 1000 °C for 1 h,
according to the literature.32 Acetic anhydride and pyridine
were dried and distilled prior to use. All other chemicals
were of reagent grade and used as received. Melting points
were determined by a Büchi 510 melting point apparatus and
are uncorrected. Infrared spectra were acquired with a FTIR
spectrometer using KBr for solid samples (range 4000-400
cm-1). 1H NMR spectra were recorded at 200 or 400 MHz and
13C NMR spectra at 100 MHz. Chemical shifts were recorded
in parts per million (ppm, d) relative to tetramethylsilane at
0.00 ppm or solvent (CDCl3 at 7.26 ppm or DMSO-d6 at 2.50
ppm for 1H NMR, and CDCl3 at 77.2 ppm or DMSO-d6 at
39.5 ppm for 13C NMR) as the internal standard.
Preparation of 3,4-O-benzylidene-D-ribonolactone (6)7
Benzaldehyde (0.58 mL, 0.57 mmol) was added
into a 25 mL round-bottomed flask followed by
D-ribonolactone 2 (85 mg, 0.57 mmol). The temperature
was raised to 50 °C for 10 min until a milky mixture was
formed. Then, 0.05 mL (1 drop) of 12 mol L-1 HCl was
added and the mixture was left reacting at 90 °C for 1.5 h.
After cooling down to 25 °C, 7 mL of Et2O was added
over the pinkish/yellowish crude mixture and the insoluble
solid was separated by filtration and washed with 3 mL
of 5% NaHCO3, 3 mL of H2O, and 3 mL of Et2O to give
6 as a white solid (94 mg, 50% yield).
Preparation of 2,3,5-tri-O-acetyl-D-ribonolactone (3)14
A mixture of D-ribonolactone 2(148 mg, 1.0 mmol),Ac2O
(1.9 mL, 20 mmol), and 13X/KCl (600 mg) was stirred at
25 °C for 3 h. The catalyst was filtered off through a short pad
of Celite® and then rinsed with 2-3 mL of EtOAc. The organic
phasewasconcentratedunderreducedpressuretogive273mg
(99% yield) of the desired product 3 as a light yellow oil.
Preparation of 5-O-acetyl-2,3-O-isopropylideneribonic-
1,4-lactone (7) and 2-O-acetyl-3,4-O-benzylidene-D-
ribonolactone (8)50
D-ribonolactone derivative 5 or 6 (0.18 mmol), Ac2O
(0.95 mL, 10 mmol), and 13X/KCl (300 mg) were stirred at
80 °C for 1 h.After cooling down to 25 °C, the mixture was
diluted with 3 mL of EtOAc and then filtered through a short
pad of celite. The filtrate was coevaporated with toluene
in the rotary evaporator to give a white solid, which was
diluted in EtOAc and filtrated again to remove traces of the
catalyst to give the desired product after evaporation of the
solvent.Yield for 7: 33 mg (99%); yield for 8: 42 mg (99%).
Recyclability of the 13X/KCl catalyst
D-Ribonolactone 2 (50 mg, 0.34 mmol),Ac2O (10 mmol)
and 13X/KCl (300 mg) were added in a conic bottle flask
coupled with a filter. The reaction was left at 25 °C under
N2 atmosphere. After 3 h, the mixture was filtered off under
a positive pressure of N2. The white solid catalyst was then
rinsed with 5 mL of EtOAc, dried, and the mixture filtered
again. The system was flushed with nitrogen for 5 min to
remove excess solvent and new reagents were added in the
flask. This process was repeated for another 2 times.
General preparation of peracetylated carbohydrates using
13X/KCl
A mixture of the carbohydrate (0.50 mmol), Ac2O
(10 mmol), and 13X/KCl (300 mg) was stirred at 80 °C
for 24 h. After cooling down to 25 °C, the catalyst was
filtered off through a short pad of Celite® and then rinsed
with 2-3 mL of EtOAc. The organic phase was concentrated
under reduced pressure to give the desired product.
Preparation of 2,3-O-isopropylidene-D-ribonolactone (5)3,4,7
D-Ribonolactone 2 (148 mg, 1.0 mmol) and acetone
(1.5 mL) were added into a 10 mL round-bottomed flask

762
Molecular Sieves Mediated Green Per-O-Acetylation of Carbohydrate Templates
J. Braz. Chem. Soc.
Pentaacetyl-D-glucose,37 pentaacetyl-D-galactose,38,39
pentaacetyl-D-mannose,40 tetraacetyl-D-xylose,41 and
tetraacetyl-D-ribose33 showed physical and spectral data in
accordance with their expected structure and by comparison
with spectral data in literature.
of potassium phosphate buffer (30 mmol L-1, pH 7). Control
experiments carried out in the absence of the enzyme
showed no reaction of compound 3.
Three-step preparation of 2,3-di-O-acetyl-D-ribonolactone
(4)
General preparation of peracetylated carbohydrates using
pyridine
D-ribose (1, 10.0 g, 67.0 mmol), sodium bicarbonate
(11.2 g, 130 mmol), and water (60 mL) were added in a
125 mL three-necked round-bottomed flask with an internal
thermometer, a pressure-equalizing addition funnel and
stirring bar. The mixture was stirred at room temperature for
15 min. Then, the flask was immersed in an ice-water bath.
Bromine (1.12 g, 70 mmol) was added to the vigorously
stirred aqueous solution through the addition funnel at a
rate such that the reaction temperature was kept under 5 °C.
After the addition, the funnel was replaced with a stopper
and the mixture stirred for another 50 min. Sodium bisulfite
(0.65 g, 6.25 mmol) was added and the orange color
solution turned transparent. Then, the aqueous solution was
transferred to a 250 mL flask and evaporated on a rotary
evaporator to give a wet slurry. Absolute EtOH (40 mL)
and toluene (10 mL) were added to the cloudy suspension
and the solvent removed by rotary evaporation to provide
a damp solid. Absolute EtOH (40 mL) was poured to the
crude and the mixture heated on a steam bath for 30 min.
The hot ethanolic suspension was filtered and the solids
rinsed with hot absolute EtOH (10 mL).After refrigerating
overnight, white crystals were formed, filtered, rinsed with
cold absolute EtOH (10 mL) followed by Et2O (10 mL) to
give, after drying under vacuum, 8.16 g of a white solid
(it is expected 35-45% of NaBr).3 Part of D-ribonolactone
from the previous procedure (2.20 g), 13X/KCl molecular
sieves (3.0 g) and Ac2O (20 mL) were stirred at 25 °C
for 3 h. The reaction was quenched with EtOAc (20 mL)
and filtered through a short pad of Celite®. The solvent
was azeotropically removed using toluene (2 × 5 mL) as
co-solvent in rotary evaporator to give 1.46 g of the crude
acylated product 3 (> 95% pure by 1H NMR) that was used
in the next step without further purification. To a mixture of
2,3,5-tri-O-acetyl-D-ribonolactone (3, 1.46 g, 4.80 mmol) in
EtOH (50 mL) was added CAL-B (730 mg) and the reaction
was left in a temperature-controlled incubator shaker
(180 rpm, 35 °C) for 6 h. Next, the supernatant solution
was decanted and the enzyme washed with MeOH (10 mL)
followed by ACN (10 mL). The combined solvents were
removed under reduced pressure to give a light yellow oil
after purification by flash column chromatography using
1:1 EtOAc/hexanes to give 570 mg of 2,3-di-O-acetyl-D-
Per-O-acetylation of natural carbohydrates catalyzed
by pyridine followed literature procedure, with slight
modifications.33 A mixture of the carbohydrate (1.0 mmol),
Ac2O (1.9 mL; 20 mmol), and pyridine (3 mL) was stirred
at 25 °C for 24 h. The reaction mixture was diluted with
5 mL of CH2Cl2 and the organic phase was washed with
1 mol L-1 HCl (3 × 5 mL), saturated NaHCO3 (3 × 5 mL),
and brine (5 mL). The solvent was dried with anhydrous
Na2SO4 and evaporated to furnish the per-O-acetylated
carbohydrate derivatives. Peracetylated carbohydrates
where characterized as above and showed physical and
spectral data in accordance with literature.37-41
Determination of the catalyst amount for the alcoholysis of 3
The optimal amount of CAL-B was determined by
reacting 2,3,5-tri-O-acetyl-D-ribonolactone (3, 0.5 mmol)
in EtOH (5.0 mL) using 18, 35, 70, 140, and 280 mg of the
biocatalyst in a temperature-controlled incubator shaker
(150 rpm, 35 °C). After 6 h, each supernatant solution was
decanted, the enzyme was washed with 3 mL of MeOH
followed by 3 mL of ACN and the solvent removed under
reduced pressure. The light yellow oil obtained was then
analyzed by 1H NMR to determine the amount of 2,3-di-O-
acetyl-D-ribonolactone (4) produced in each case (Table 5).
Enzyme catalyzed solvolysis in different alcohols
Experiments of biocatalyzed alcoholysis were
performed by adding CAL-B (140 mg) to a suspension
of the 2,3,5-tri-O-acetyl-D-ribonolactone (3, 50 mg,
0.18 mmol) in 5.0 mL of an alcohol and shaking the
resulting mixtures in a temperature-controlled incubator
shaker (150 rpm, 35 °C). After 0.5, 1.0, 2.0, 4.0, and 6.0 h
(Table 6), biotransformations were stopped by filtering
off the enzyme and washing the biocatalyst with 3 mL of
MeOH followed by 3 mL of ACN. The recyclability of
CAL-B was determined by applying the previously used
supported enzyme in a new cycle.
Experiments involving the hydrolysis of 3 catalyzed
by CAL-B were carried out according to the procedure
described above, replacing the alcohol by the same volume
25
ribonolactone (4). Overall yield 14% from 1. [a]D +5.5
(c 5, EtOH); IR (film) nmax/cm-1 3495 (OH), 2950, 1796

Vol. 26, No. 4, 2015
Cardozo et al.
763
1
(lactone), 1750 (ester), 1377, 1247, 1083; H NMR
(400 MHz, CDCl3) d 5.83 (d, 1H, J 6.3 Hz, H-2), 5.54 (d,
J 6.3 Hz, 1H, H-3), 4.57-4.59 (m, 1H, H-4), 4.01 (dd, 1H,
J 12.1 Hz, CH2), 3.95 (d, 1H, J 12.1 Hz, CH2), 2.16 (3H, s),
2.13 (3H, s); 13C NMR (75 MHz, CDCl3) d 20.2, 20.5, 61.4,
67.1, 70.6, 83.5, 169.4, 170.0, 171.5; ESI-HRMS (CH3OH)
calcd.: C9H13O7 [M + H]+: 233.0656; found: 233.0662.
11. Boojamra, C. G.; Cannizzaro, C.; Chen, J. M.; Chen, X.;
Cho, A.; Chong, L. S.; Fardis, M.; Jin, H.; Hirschmann, R. F.;
Huang,A. X.; Kim, C. U.; Kirschberg, T.; Lee, C. P.; Lee, W.A.;
Mackman, R. L.; Markevitch, D.Y.; Oare, D. A.; Prasad, V. K.;
Pyun, H.-J.; Ray,A. S.; Sherlock, R.; Swaninathan, S.; Watkins,
W. J.; Zhang, J.; US pat. 0288053, 2011.
12. Stefko, M.; Slavetínská, L.; Klepetářová, B.; Hocek, M.; J. Org.
Chem. 2010, 75, 442.
Supplementary Information
13. Kubelka, T.; Slavetínská, L.; Hocek, M.; Eur. J. Org. Chem.
2012, 4969.
Supplementary information (1H NMR for all
compounds synthesized and 13C NMR for compounds 4,
5, 7, 8, and peracetylated carbohydrates) is available free
of charge at http://jbcs.sbq.org.br as PDF file.
14. Sá, M. M.; Silveira, G. P.; Castilho, M. S.; Pavão, F.; Oliva, G.;
Arkivoc 2002, 8, 112.
15. Adinolfi, M.; Barone, G.; Iadonisi, A.; Schiattarella, M.;
Tetrahedron Lett. 2003, 44, 4661.
16. Kumareswaran, R.; Pachamuthu, K.;Vankar,Y. D.; Synlett 2000,
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Acknowledgements
17. Bhaskar, P. M.; Loganathan, D.; Synlett 1999, 129.
18. http://www.inchem.org/documents/iarc/vol77/77-16.html
accessed in December 2014.
The authors wish to thank Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and
Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) for fellowships and scholarships.
M. M. S. is grateful to CNPq for a research fellowship.
G. P. S. is grateful to CNPq and CAPES for a PVE - science
without borders (096/2013) and CAPES/UDELAR bilateral
program - projetos conjuntos de pesquisa (049/2013).
Financial support from INCT-Catalysis/CNPq (Brazilian
Research Council) is also gratefully acknowledged.
19. Berry, D. J.; DiGiovanna, C. V.; Metrick, S. S.; Murugan, R.;
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23. Dutra, L. G.; Saibert, C.; Vicentini, D. S.; Sá, M. M.; J. Mol.
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