Regioselective acylation of d-ribono-1,4-lactone catalyzed by lipases

Article abstract of DOI:10.1016/j.procbio.2010.10.007

Lipases from ten different sources and two mycelium-bound lipases isolated from Amazonian fungi were screened as biocatalysts in the acylation reaction of d-ribono-1,4-lactone with a variety of acyl donors in non-aqueous media. Several reaction parameters were evaluated including the type and amount of enzyme, acyl donor, and organic solvent, as well as the influence of water and the recyclability of the catalyst. When Candida antarctica lipase (CAL-B) was used, the acylation was highly regioselective and the corresponding 5-acyl-d-ribono-1,4-lactones were observed as the sole product. The best conversion (>99%) into 5-acetyl-d-ribono-1,4-lactone was obtained through the combination of vinyl acetate as the acetyl donor and 10 mg (100 U) of CAL-B in dry acetonitrile after 24 h. However, lipases from Burkholderia cepacia (PSL-C and PSL-D), Pseudomonas fluorescens (AK) and Thermomyces langinosus (Lipozyme TL-IM) gave mixtures of mono-, di- and tri-acetylated products in lower conversions. CAL-B maintained its catalytic activity during five cycles of repeated use when decanoic and dodecanoic acids were employed as acyl donors in the acylation of d-ribono-1,4-lactone.

Full text of DOI:10.1016/j.procbio.2010.10.007

Process Biochemistry 46 (2011) 551–556
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Regioselective acylation of d-ribono-1,4-lactone catalyzed by lipases
Damianni Sebrão, Marcus M. Sá, Maria da Grac¸ a Nascimento∗
Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2010
Received in revised form 8 October 2010
Accepted 19 October 2010
Lipases from ten different sources and two mycelium-bound lipases isolated from Amazonian fungi were
screened as biocatalysts in the acylation reaction of d-ribono-1,4-lactone with a variety of acyl donors
in non-aqueous media. Several reaction parameters were evaluated including the type and amount of
enzyme, acyl donor, and organic solvent, as well as the influence of water and the recyclability of the
catalyst. When Candida antarctica lipase (CAL-B) was used, the acylation was highly regioselective and
the corresponding 5-acyl-d-ribono-1,4-lactones were observed as the sole product. The best conversion
(>99%) into 5-acetyl-d-ribono-1,4-lactone was obtained through the combination of vinyl acetate as the
acetyl donor and 10 mg (100 U) of CAL-B in dry acetonitrile after 24 h. However, lipases from Burkholderia
cepacia (PSL-C and PSL-D), Pseudomonas fluorescens (AK) and Thermomyces langinosus (Lipozyme TL-IM)
gave mixtures of mono-, di- and tri-acetylated products in lower conversions. CAL-B maintained its
catalytic activity during five cycles of repeated use when decanoic and dodecanoic acids were employed
as acyl donors in the acylation of d-ribono-1,4-lactone.
Keywords:
5-Acyl-d-ribono-1,4-lactones
Lipases
Regioselective acylation
Sugar fatty acids
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
source of the lipase, as well as many of the reaction conditions, is
responsible for dramatic differences in the reaction outcome, and
Carbohydrates are valuable sources for the production of syn-
thetic compounds of general relevance [1–3]. For example, sugar
fatty acid esters are an important class of biodegradable non-ionic
surfactants with widespread application in the food, cosmetic and
pharmaceutical industries [4]. The acylation of an alcohol to pro-
duce an ester is amongst the most fundamental organic reactions,
and acylation of monosaccharides is often involved in the multi-
step synthesis of complex carbohydrates [5–7]. While the acylation
of simple alcohols can be readily accomplished by base-catalyzed
transesterification under relatively simple conditions, the regios-
elective synthesis of acyl esters of sugars is much more difficult
and unpredictable due to the presence of multiple hydroxyl groups.
Therefore, an efficient multistep synthesis of carbohydrate deriva-
tives frequently requires the introduction of protective groups and
their subsequent removal [8–10].
On the other hand, enzymatically catalyzed sugar fatty acid
esterification reactions are, in general, reasonably specific and
the regioselective acylation of several carbohydrates with hydro-
lases such as lipases, esterases and proteases has been reported
[11–14]. Lipases (EC 3.1.1.3) are the most used for this purpose,
with recent reports describing efficient protocols for the selective
lipase-catalyzed acylation of monosaccharide derivatives and poly-
meric prodrugs of 5-fluorouridine [15]. It is also known that the
these parameters can be fine tuned in order to generate the desired
regioisomeric product.
Chemical transformations which do not require harmful
reagents or complex purification techniques and do not gener-
ate toxic waste represent a fundamental target of contemporary
organic synthesis [16–18]. The high selectivity of enzymes can sim-
plify industrial processes by increasing the chemical yields and
reducing the generation of by-products, and biocatalysis is thus
an important tool in the development of green chemistry [19,20].
d-Ribono-1,4-lactone (1) is an inexpensive and abundant sugar
derivative that is commercially available from renewable resources
[21,22]. Because of its high functionality with contiguous chiral cen-
ters, 1 has been widely used as a versatile chiral building block
for the construction of a variety of natural products [23–25]. In
principle, the acetylation of d-ribono-1,4-lactone (1) may lead to
seven distinct acetylated products (three mono-, three di- and one
triacetylated derivatives, Scheme 1). However, many other unex-
pected products can also be formed in one simple reaction involving
carbohydrates due to the possible presence of different species
co-existing in equilibrium under certain reaction conditions and
also to processes ranging from rearrangements to functional group
migration [26–28].
As part of our research interest in lipase-catalyzed transfor-
mations [29–31], we studied the regioselective acylation of the
primary and secondary hydroxyl groups of d-ribono-1,4-lactone
(1) catalyzed by lipases from different sources under a variety of
reaction conditions, and the results are presented herein.
∗
Corresponding author. Tel.: +55 48 37216844; fax: +55 48 37216850.
E-mail address: graca@qmc.ufsc.br (M.G. Nascimento).
1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.10.007

552
D. Sebrão et al. / Process Biochemistry 46 (2011) 551–556
4
1
24 h. The solid product that precipitated out was separated by filtration (together
O
O
lipases or mycelium
R1O
O
O
HO
5
with the insoluble enzyme). Re-dissolution of the cake in a 1:1 ethanol/ethyl ether
mixture followed by filtration to separate the catalyst gave 14 as a crystalline solid
after evaporation of the solvent and recrystallization in ethanol/ethyl ether (68%
acetyl donor
solvent
35 ºC, 150 rpm
3
2
HO
OH
R2O
OR3
1
yield); m.p. 123–125 ◦C; IR: 3497, 3304, 1765, 1754, 1244 and 1160 cm−1 1H NMR
;
(DMSO-d6): ı 0.85 (m, 3H), 1.24 (m, 16H), 1.49 (m, 2H), 2.32 (t, 7.2 Hz, 2H), 4.11–4.20
(m, 2H), 4.24 (dd, 3.6 Hz, 12.0 Hz, 1H), 4.40–4.45 (m, 2H), 5.60 (d, 3.6 Hz, 1H, D2O
exchange), 5.92 (d, 7.6 Hz, 1H, D2O exchange); 13C NMR (DMSO-d6): ı 14.6 (CH3),
22.8 (CH2), 25.0 (CH2), 29.1 (CH2), 29.2 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2),
29.7 (CH2), 32.0 (CH2), 33.9 (CH2), 63.6 (CH2), 69.0 (CH), 69.5 (CH), 82.6 (CH), 173.0
(C O), 176.0 (C O). Anal. calcd. for C17H30O6: C, 61.8; H, 9.2. Found: C, 60.7; H, 9.4.
R1 R2 R3
R1 R2 R3
Ac Ac
Ac Ac
Ac Ac Ac
2
3
4
5
H
H
Ac
H
6
7
8
H
H
Ac
Ac Ac
H
H
Ac
H
H
3. Results and discussion
Scheme 1. Acetylation of d-ribono-1,4-lactone (1) catalyzed by lipases.
3.1. Screening of enzymes
2. Experimental
In the first approach, ten commercially available lipases, in free
or immobilized forms, and two mycelium-bound lipases isolated
from native Amazon plants (Brazil) [32] were screened for their
efficiency in the acetylation of lactone 1 with vinyl acetate. The
relative composition of acetylated products was based on analysis
of the 1H NMR spectra of the crude reaction products after certain
time periods (6 and 24 h) as shown in Table 1.
From the data presented in Table 1 it is clear that the conver-
sion degree and product distribution were dependent on the source
of the biocatalyst. Poor activities were observed for PSL, F-AP15,
CRL, ROL, ANL lipases and UEA 53 mycelium, with no product being
detected even after 72 h reaction (results not shown). On the other
hand, much better reaction profiles were obtained with lipase B
from Candida antarctica (CAL-B) as the biocatalyst, furnishing the
5-monoacetylated derivative 5 as the sole product in a quantita-
tive conversion (>99%) after 24 h, although high conversions were
also observed in shorter times (Table 1, entry 1). With the use of
Lipozyme TL-IM, a low-cost biocatalyst previously employed for the
regioselective acylation of 5-fluorouridine analogs [15], the conver-
sions into the monoacetylated products 3 and 5 were slow and not
regioselective under the standard conditions (10 mg catalyst, entry
2). However, the conversion to 5 could be reasonably increased to
85% with complete regioselectivity (5:3 ratio >99:1) using larger
amounts of the catalyst (entry 3).
For the other lipases studied (Table 1, entries 4, 5 and 7),
very poor regioselectivities were observed under our standard
conditions or in the presence of larger amounts of the catalyst
(entries 6 and 8), which accelerates the reaction at the expense
of the selectivity. In all but one case (entry 7), the product
mixture was composed of 5-O-acetyl-d-ribonolactone (5) as the
main component, together with 3-O-acetyl- (3), 2,5-O-diacetyl-
(6), 3,5-O-diacetyl- (7) and/or 2,3,5-O-triacetyl-d-ribonolactone (8)
as recurrent minor by-products. The competitive formation of
mono- and di-acetylated compounds was inferred from the NMR
spectra of the crude products since it is relatively easy to differ-
entiate between a carbinolic (CHOH) hydrogen nucleus and its
O-acetylated derivative (CHOAc). Also, the spectral data are con-
sistent with the expected structure and with the data for model
compounds [10,15,25,33,34].
2.1. Materials
All chemicals were of reagent grade and were used as received. Candida
antarctica lipase B (CAL-B, Novozym 435, 10,000 PLU/g) and Thermomyces langi-
nosus (Lipozyme TL-IM, 250 IUN/g) were donated by Novozymes A/S. Lipases from
Burkholderia cepacia (PSL 30,000 U/g; PSL-C “Amano” I, 1638 U/g; and PSL-D “Amano”
I, 744 u/g), Pseudomonas fluorescens (AK, 26,600 U/g) and Rhizopus oryzae (F-AP15,
150 u/mg) were donated by Amano Enzymes Inc. Candida rugosa lipase (CRL, ≥700
unit/mg solid) was purchased from Sigma–Aldrich. Native lipases from Aspergillus
niger AC-54 (ANL, 19.4 U/mL) and Rhizopus oligosporus (ROL, 14.9 U/mL) were kindly
donated by Professor Patrícia de Oliveira Carvalho from Universidade de São Fran-
cisco (USF), Braganc¸ a Paulista, SP, Brazil and were isolated from microorganisms
collected in the region of Bueno Brandão, MG, Brazil. The mycelium-bound lipases
isolated from Amazonian fungi UEA 53 (Astrocaryum aculeatum) and UEA 115
(Amazon wood) were kindly donated by Professor Sandra Patrícia Zanotto from Uni-
versidade Estadual do Amazonas (UEA), Manaus, AM, Brazil. d-Ribono-1,4-lactone
and hexanoic, octanoic, decanoic, gallic, mandelic, phenylacetic and octadecanoic
acids were purchased from Sigma–Aldrich. Butanoic, dodecanoic and hexadecanoic
acids, ethyl acetate, ethyl acetoacetate and acetic anhydride were provided by Vetec.
Vinyl acetate, isopropenyl acetate and tetradecanoic acid were obtained from Fluka
Chemika. Acetic and propanoic acids were provided from Reagen. Benzoic acid,
glycine, d,l-alanine and l-cysteine were obtained from Merck.
2.2. Analytical methods
Infrared spectra were acquired with a Perkin-Elmer FTIR 1600 spectrometer
using KBr for solid samples (range 4000–400 cm−1). 1H NMR and 13C NMR spec-
tra were recorded at 400 and 100 MHz, respectively, with a Varian 400 Mercury
Plus spectrometer, using DMSO-d6 as the solvent and TMS as the internal standard.
Elemental analyses were conducted in a Carlo Erba CHNS EA-1110 instrument at
Central Analítica, UFSC, Florianópolis, SC, Brazil.
2.3. General enzymatic procedure for the acylation of d-ribono-1,4-lactone (1)
The selected lipase was added to a solution containing the lactone 1 (0.5 mmol)
and an acyl donor (1.5 mmol) in 10.0 mL of an anhydrous solvent and the reaction
mixture was incubated in a shaker at 35 ◦C and 150 rpm. After the allotted time for
each reaction, 1.0 mL aliquots were withdrawn and the reaction was stopped by fil-
tering off the lipase. Conversions (%) were determined by 1H NMR by comparing the
signals of the initial lactone 1 with the corresponding signals of the acylated prod-
ucts. Control experiments were also conducted without lipases (free or immobilized)
under similar reaction conditions, and no product was formed.
2.4. Synthesis of 5-O-acetyl-d-ribono-1,4-lactone (5) catalyzed by CAL-B
The reaction was initiated by dissolving d-ribono-1,4-lactone
1 (74.0 mg,
0.5 mmol) and vinyl acetate (0.14 mL, 1.5 mmol) in anhydrous acetonitrile (10.0 mL)
followed by the addition of CAL-B (10.0 mg). The mixture was shaken at 35 ◦C and
150 rpm for 24 h. The reaction was stopped by filtering off the lipase. Finally, solvent
was evaporated and 5 was obtained as a white solid after recrystallization in ace-
tone (94% yield); m.p. 140-143 ◦C (lit.10 148 ◦C); IR: 3476, 3289, 1761, 1749, 1429
Finally, interesting results were found for the UEA 115
mycelium, which catalyzed the regioselective 5-monoacetylation
of ribonolactone 1 to produce 5, in spite of the slow reaction rate
(41% conversion after 72 h) using a large amount of catalyst (Table 1,
entries 9 and 10).
Thus, with regard to the selectivity observed for CAL-B and
Lipozyme TL-IM, the preferential acetylation at the less-hindered
primary 5-hydroxy group leading to the regioselective formation
of 5-acetylated product 5 is in agreement with similar studies in
the literature [13,15,34–38], although this was not the case for all
of the other enzymes tested. Considering the above results, CAL-B
was selected for the following studies.
and 1385 cm−1 1H NMR (DMSO-d6): ı 2.03 (s, 3H), 4.10-4.17 (m, 2H), 4.24 (dd,
;
3.6 Hz, 12.4 Hz, 1H), 4.38-4.43 (m, 2H), 5.57 (d, 4.0 Hz, 1H, D2O exchange) and 5.87
(d, 7.6 Hz, 1H, D2O exchange); 13C NMR (DMSO-d6): ı 21.1 (CH3), 63.6 (CH2), 68.9
(CH), 69.4 (CH), 82.7 (CH), 171.0 (C O), 177.0 (C O). Anal. calcd. for C7H10O6: C,
44.2; H, 5.3. Found: C, 44.1; H, 5.3.
2.5. Synthesis of 5-O-lauryl-d-ribono-1,4-lactone (14) catalyzed by CAL-B
Anhydrous acetonitrile (10.0 mL) was added to a mixture of d-ribono-1,4-
lactone
1 (459 mg, 3.1 mmol) and lauric acid (621 mg, 3.1 mmol) followed by
addition of CAL-B (70.0 mg). The final mixture was stirred at 150 rpm and 35 ◦C for

D. Sebrão et al. / Process Biochemistry 46 (2011) 551–556
553
Table 1
Acetylation of 1 with vinyl acetate using different biocatalysts
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
O
O
O
O
O
O
O
O
O
OH
HO
OH
HO
O
O
OH
O
O
5
6
7
8
3
a
.
Lipase
Mcat (mg)
Conversion (%)b
6 h
24 h
3
5
6
7
3
5
6
7
c
c
1
2
3
4
5
6
7
8
9
10
CAL-B
10
10
40
10
10
60
10
40
10
40
–
7
85
16
–
–
nd
–
–
13
–
nd
–
–
–
–
5
–
8
>99
24
85
12
28
46
13
29
8
–
–
–
–
–
–
8
16
23
–
11
–
–
Lipozyme TL-IM
Lipozyme TL-IM
AK
PSL-C “Amano” I
PSL-C “Amano” Ie
PSL-D “Amano”
PSL-D “Amano”
nd
8
14
14
7
nd
–
nd
ndd
10
19
32
10
nd
–
nd
6
11
14
–
nd
–
nd
–
17
–
15
23
–
11
17
–
7
–
Mycelium UEA 115
Mycelium UEA 115
nd
nd
–
41f
–
a
Reaction conditions: 1 (0.5 mmol), vinyl acetate (1.5 mmol), lipase or mycelium, acetonitrile (10 mL), 35 ◦C, 150 rpm.
b
c
d
e
f
Determined by 1H NMR (400 MHz, DMSO-d6).
Negligible conversion (<1%).
Not determined.
Triacetylated product 8 (14%) was also obtained after 24 h reaction.
Conversion after 72 h reaction.
3.2. Effect of organic solvent and presence of water
It is important to observe that all the solvents suitable for
the regioselective acetylation of 5 (Table 2, entries 1–3) possess
reduced toxicity and also have low boiling points, which make
the work-up and purification steps easier in comparison with
other methodologies that employ expensive and high-boiling point
chemicals, such as pyridine, dimethyl sulfoxide or DMF.
It is well established that enzyme activity can be dramatically
affected by the choice of the organic solvent, and good correlations
between the reaction rate and the polarity of the solvent have been
achieved [39–41]. Log P (logarithm of the partition coefficient of the
solvent for the standard octanol/water two-phase system) is the
most useful parameter to classify the solvents for biocatalytic reac-
tions. Accordingly, solvents with log P ≤ 2 are more hydrophilic and
tend to strip away the water molecules present on the surface of the
enzyme, reducing the catalytic activity. Conversely, solvents with
high log P (≥4) are hydrophobic and stabilize the enzymes, being
the most suitable for biocatalytic processes [39]. In order to study
the solvent effect, a series of polar aprotic solvents were used for
the CAL-B-catalyzed acetylation reaction of lactone 1, which is not
soluble in solvents of low polarity. Acetonitrile, acetone, tetrahy-
drofuran (THF), dioxane and N,N-dimethylformamide (DMF) were
selected for the acetylation of 1 with vinyl acetate (Table 2).
The data given in Table 2 show that there was no clear rela-
tionship between log P and the conversion degrees. Using the less
polar solvents (log P ≥ −0.49) such as acetone, THF and acetoni-
trile, the 5-monoacetylated derivative 5 was selectively obtained
with conversion degrees ranging from 61% to 72% (entries 1–3). On
the other hand, in the case of dioxane (log P = −1.1) there was not
only a considerably lower conversion to the expected product 5
but also a decrease in the regioselectivity (entry 4), whereas DMF
(log P = −1.0) completely inhibited the formation of any product
(entry 5).
Another parameter which needs to be evaluated is the water
content, which plays an important role in controlling the enzyme
performance in organic media. It is well described that lipases pos-
sess a water monolayer at their surface, or clusters around the
charged groups of the protein, which maintains the native con-
formation even in organic solvents. The optimal amount of water
required depends on several parameters, such as the type of sol-
vent, substrate, and solid support, the polarity of the enzyme active
site, and the reaction conditions [42]. However, the presence of
water in the reaction medium can decrease the conversion degrees
in lipase catalyzed acylations, shifting the equilibrium towards
ester hydrolysis to give the corresponding carboxylic acids [43].
Therefore, CAL-B-catalyzed acetylation of 1 with vinyl acetate
was also studied with the addition of 27.0 ␮L (3 equiv.) of water. The
influence of the reaction time was then evaluated in this reaction
(Fig. 1). While the degree of conversion to the product 5 (∼50% after
24 h) was lower than that obtained using dry acetonitrile (>99%,
24 h), the regioselectivity of the process was entirely maintained.
The results presented in Fig. 1 also show that the conversion to 5
in dry acetonitrile rapidly increased in the first 6 h (up to 85%), and
from this point onwards it formed a plateau reaching a quantitative
conversion to 5 after 24 h. Thus, considering the results obtained
herein, dry acetonitrile as the solvent and a reaction time of 6 h
were selected for the subsequent studies involving the acetylation
of ribonolactone 1.
Table 2
Solvent effects on the CAL-B-catalyzed acetylation of 1.
Entry
Solvent
Log Pa
Conversion to 5 (%)b
1
2
3
4
5
Acetonitrile
THF
Acetone
Dioxanec
DMF
−0.33
−0.49
−0.23
−1.1
72
68
61
44
<1
3.3. Effect of CAL-B loading
The influence of the amount of CAL-B on the regioselective
acetylation of 1 with vinyl acetate in dry acetonitrile was then eval-
uated, in the range of 0–50 mg (0–500 Units). The results, presented
in Fig. 2, show that high conversions to the 5-monoacetylated prod-
uct 5 were achieved with as little as 10 mg (100 U) CAL-B in 6 h.
−1.0
a
Ref. [39].
b
c
Determined by 1H NMR (400 MHz, DMSO-d6).
Mixture of compounds: 5 (44%), 3 (9%) and 7 (6%).

554
D. Sebrão et al. / Process Biochemistry 46 (2011) 551–556
Table 3
100
75
50
25
0
Influence of acetyl donor on the acetylation of 1.
Acetyl donor
Conversion (%)a
3:1b,c
200:1b,d
1
2
3
4
5
Vinyl acetate
Isopropenyl acetate
Ethyl acetate
Acetic anhydride
Acetic acid
85
28
23
52
<5%
>99
75
53
16e
<5%
a
b
c
Determined by 1H NMR (400 MHz, DMSO-d6).
Molar ratio of the acyl donor to the substrate 1.
Reaction conditions: 1 (0.5 mmol), acyl donor (1.5 mmol), acetonitrile (10 mL),
CAL-B (10 mg), 35 ◦C, 150 rpm, 6 h.
d
Reaction conditions: 1 (0.5 mmol), acyl donor (100 mmol), CAL-B (10 mg), 35 ◦C,
150 rpm, 6 h.
e
Mixture of products: 5 (16%), 3 (32%) and 7 (27%).
0
4
8
12
16
20
24
Time (h)
after 6 h reaction (entry 3). Acetic anhydride was also studied as
the acetyl donor, but its more pronounced reactivity (even in the
absence of any catalyst) [44] precluded its use in great excess due
to the competitive formation of acetylated by-products 3 and 6
(entry 4). Nevertheless, the method employing a combination of
acetic anhydride/1 (3:1) in CH3CN reestablished the regioselectiv-
ity towards the exclusive formation of 5 in moderate conversion.
As expected, acetic acid was very ineffective as the acyl donor and
no detectable product was formed under the test conditions (entry
5).
It has been stated that some carboxylic acids, such as acetic and
propionic, act as potent inhibitors of lipase activity by removing
its aqueous micro-layer and by altering the pH in the catalytic
site, contributing to the observed decrease in enzyme activity
[45].
Due to the biological importance associated with carbohy-
drate fatty acid esters [4,46], the CAL-B-catalyzed acylation of
ribonolactone 1 was extended to a series of saturated carboxylic
acids (Scheme 2). The results showed the total regioselectivity of
CAL-B-catalyzed acylations, providing exclusively the correspond-
ing 5-acylated sugar derivatives. Low to good conversions were
obtained depending on the alkyl chain length of the acyl donor
(Fig. 3). As previously discussed, no product was formed when
acetic acid was employed. Better results were observed when pro-
pionic and butyric acids were used, with conversions of 28% and 38%
being obtained for the corresponding esters 9 and 10, respectively.
Higher conversion degrees were obtained for linear carboxylic acids
with six, eight, ten, twelve and fourteen carbons, giving the 5-acyl
derivatives 11–15 in the range of 50–67%. However, a decrease
in the conversion degrees was observed when palmitic (C16) and
stearic (C18) acids were employed, forming the corresponding
products 16 and 17 in 41% and 46%, respectively. In these cases, the
lower conversion degrees may be due to stereo-electronic effects
as well as a reduced solubility of acyl donors in acetonitrile. These
results are in agreement with previously reported data, wherein
CAL-B was described as highly active for short and medium chain
length carboxylic acids and esters, but of decreased activity for
long-chain fatty acids and esters [31,47–49].
Fig. 1. Influence of time on the regioselective acetylation of 1 in dry acetoni-
trile (ꢀ) and in acetonitrile + water (27.0 ␮L; 1.5 mmol) (᭹). Reaction conditions: 1
(0.5 mmol), vinyl acetate (1.5 mmol), CAL-B (10 mg, 100 Units), acetonitrile (10 mL),
35 ◦C, 150 rpm.
Therefore, the substrate/catalyst ratio employed in the subsequent
experiments was set at 0.5 mmol/10 mg.
3.4. Influence of amount and type of acyl donor
Next, the type and relative amount of acyl donor (carboxylic
acid, anhydride or ester) were evaluated for the regioselective acy-
lation of lactone 1 using CAL-B as the biocatalyst. Two methods
were designed for carrying out the transformations: in the first,
a 3:1 molar ratio of acyl donor/substrate was used with acetoni-
trile as the solvent; in the second, a large excess of the acyl donor
(200 equiv.) was employed as both the reactant and solvent. The
observed conversions to the 5-monoacetylated product 5 are given
in Table 3.
Good conversion degrees and regioselective formation of 5 were
obtained for most of the acetyl donors screened. The highest con-
version degree was achieved with vinyl acetate, for both methods
studied (Table 3, entry 1). On the other hand, isopropenyl acetate
is only useful in high excess (entry 2). Interestingly, using ethyl
acetate, which is of very low cost, as the reactant/solvent gave
an acceptable 53% conversion to the 5-monoacetyl derivative 5
100
75
50
25
0
Therefore, the variety of acyl donors that are compatible with
CAL-B makes this biocatalyst a versatile tool for regioselective acy-
lations. Moreover, using carboxylic acids as the acyl donor the only
O
O
O
O
0
100
200
300
400
500
O
O
HO
R
O
R
OH
CAL-B
CH3CN, 25 o
CAL-B (Units)
1
HO
OH
HO
OH
C
Fig. 2. Influence of CAL-B units on the acetylation of 1. Reaction conditions: 1
(0.5 mmol), vinyl acetate (1.5 mmol), CAL-B (0–50 mg; 0–500 Units), dry acetonitrile
(10 mL), 35 ◦C, 150 rpm, 6 h.
Scheme 2. Regioselective acylation of d-ribono-1,4-lactone (1) catalyzed by CAL-B.

D. Sebrão et al. / Process Biochemistry 46 (2011) 551–556
555
100
80
60
40
20
Fig. 3. Effect of chain length on the regioselective acylation of 1. Reaction condi-
tions: 1 (0.5 mmol), acyl donor (1.5 mmol), acetonitrile (10 mL), CAL-B (10 mg), 35 ◦C,
150 rpm, 24 h.
0
1
2
3
4
5
Utilizations
by-product generated was water, which is in line with a clean and
environmentally friendly processes [16–20]. However, attempts to
acylate ribonolactone 1 with functionalized carboxylic acids such
as benzoic, phenylacetic, galic and mandelic acids, as well as some
amino acids including glycine, d,l-alanine and l-cysteine, under the
experimental reaction conditions described above, did not lead to
any detectable product in the crude reaction after 48 h. One possi-
ble reason is that the presence of polar groups negatively influences
the reaction profile, since it is known that CAL-B is not a suitable
catalyst for the esterification of sugars with unsaturated or ary-
laliphatic acids like cinnamic acid and its derivatives, benzoic acids,
phenolic derivatives and ␣-substituted carboxylic acids, with some
exceptions [50].
Fig. 4. Effect of catalyst reuse on the acylation of 1 with vinyl acetate (ꢀ) in 6 h reac-
tion, and with decanoic (᭹) and dodecanoic (ꢁ) acids in 24 h. Reaction conditions:
1 (0.5 mmol), acyl donor (1.5 mmol), CAL-B (10 mg), acetonitrile (10.0 mL), 35 ◦C,
150 rpm.
tion medium by filtration and washed with acetonitrile in order to
remove any substrate or product retained in the catalyst surface.
In the next step, the substrates and the recuperated catalyst were
added to a new reaction medium and at the end of each reaction,
the conversion degrees were determined. The results are presented
in Fig. 4.
An interesting way to install a functional group moiety in the
5-position of the ribonolactone framework is through a transes-
terification reaction using an excess of ethyl acetoacetate as the
acyl donor. A three-fold excess of ethyl acetoacetate led to the 5-
acetoacetyl-d-ribonolactone derivative 18 in a conversion degree
of 28% after 24 h. However, the conversion could be increased to
50% by employing a considerable excess (100:1) of ethyl acetoac-
etate (Scheme 3). Although the conversion was relatively low, given
that this one-step transformation is clean and offers simplicity
it compared very favorably with the previous preparation of 18
from 1 which involves three steps (protection/deprotection pro-
tocols), gave a low overall yield, and requires toxic reagents such
as diketene, pyridine and benzene [51]. Since the 5-acetoacetyl-
d-ribonolactone derivative 18 is a precursor of biologically active
compounds, this CAL-B-catalyzed transesterification reaction could
have useful applications in synthetic organic chemistry, and further
studies are underway.
With the use of vinyl acetate the conversion to 5 decreased
after each reutilization, reaching a value of 15% in the fifth cycle.
Although vinyl acetate was shown to be a suitable acetyl donor
for the acetylation of 1, the presence of significant amounts of
acetaldehyde (generated as the by-product in the transesterifica-
tion reaction) led to inactivation of the lipase by the condensation
reaction with essential amino groups of the enzyme to give Schiff
bases [52], thus preventing the application of the catalyst for con-
secutive cycles. Another possible cause of enzyme deactivation
with vinyl acetate as the acyl donor could be related to its slow
hydrolysis generating small amounts of acetic acid, which is also
harmful to the biocatalyst as discussed above.
However, the use of decanoic acid as the acyl donor led to
the maintenance of a reasonable level of catalytic activity after
each reutilization and the conversion degrees were in the range
of 57–39% after five cycles. Dodecanoic acid gave slightly better
conversions after each batch, in the range of 64–53%. Besides gener-
ating water as the by-product, these long-chain carboxylic acids are
not toxic to the enzymes and, therefore, can be employed without
any additional precautions.
3.5. Recyclability of the catalyst
The reusability of immobilized enzymes is an important prop-
erty of any catalyst and can determine the economic viability of
a biosynthetic process. In this regard, CAL-B was employed in five
subsequent cycles in the acylation of 1 with vinyl acetate, decanoic
and dodecanoic acids under the experimental conditions described
above for each acyl donor (Table 3 and Fig. 3). At the end of
each batch, the immobilized lipase was removed from the reac-
4. Conclusions
The results showed that CAL-B was the most versatile bio-
catalyst for the regioselective acylation of d-ribono-1,4-lactone
(1). However, many parameters influence the reaction outcome,
including the source of the enzyme, the nature of the acyl donor
and the organic solvent, the amount of biocatalyst and the pres-
ence of water. CAL-B could be reused in at least five cycles without
a significant loss in the activity employed decanoic and dodecanoic
acids as acyl donors and producing water as the sole by-product.
In contrast to the multistep chemical process, the enzymatic acy-
lation of d-ribono-1,4-lactone furnished, in most cases, only the
5-acylated derivative under mild conditions.
O
O
O
O
O
O
O
O
HO
O
OEt
CAL-B
18
CH3CN, 25 o
C
1
HO
OH
HO
OH
Scheme 3. Regioselective acylation of d-ribono-1,4-lactone (1) with ethyl acetoac-
etate catalyzed by CAL-B.

556
D. Sebrão et al. / Process Biochemistry 46 (2011) 551–556
ribono-1,4-lactone and 2-deoxy-d-ribono-1,4-lactone. Tetrahedron 1993;49:
Acknowledgements
349–62.
[25] Taylor CM, Barker WD, Weir CA, Park JH. Toward a general strategy for
This study was supported by Universidade Federal Santa
Catarina (UFSC-Brazil), Conselho Nacional de Desenvolvimento
Científico e Tecnoloógico (CNPq-Brazil) and INCT-Catalysis, which
provided financial support and scholarships (M.G.N., M.M.S. and
D.S.). We also thank Amano Pharmaceutical Co. (Japan), Novozymes
(Brazil) and Professor Patrícia O. Carvalho from Universidade de
São Francisco (USF-Brazil) for the donation of lipases, and Profes-
sor Sandra P. Zanotto from Universidade Estadual do Amazonas
(UEA-Brazil) for the donation of mycelia.
the synthesis of 3,4-dihydroxyprolines from pentose sugars.
2002;67:4466–74.
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