Regioselective preparation of thiamphenicol esters through lipase-catalyzed processes

Article abstract of DOI:10.5935/0103-5053.20140069

The lipase-catalyzed synthesis of thiamphenicol derivatives has been studied through complementary acylation and hydrolytic approaches, finding Candida antarctica lipase B as the most efficient biocatalyst for the selective modification of both thiampheni

Full text of DOI:10.5935/0103-5053.20140069

http://dx.doi.org/10.5935/0103-5053.20140069
J. Braz. Chem. Soc., Vol. 25, No. 6, 987-994, 2014.
Printed in Brazil - ©2014 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
Regioselective Preparation of Thiamphenicol Esters Through Lipase-Catalyzed Processes
Marcos R. da Silva,a,b Tasso G. C. Montenegro,a,b Marcos C. de Mattos,*,b
Maria da Conceição F. de Oliveira,b Telma L. G. de Lemos,b Gonzalo de Gonzalo,a
Iván Lavandera,a Vicente Gotor-Fernándeza and Vicente Gotor*,a
aDepartamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de
Asturias, Universidad de Oviedo, c/ Julián Clavería 8, 33006, Oviedo, Spain
bDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará,
60451-970 Fortaleza-CE, Brasil
A síntese de derivados do tianfenicol, catalisada por lipases, foi investigada através da realização
das reações de acilação e de hidrólise, como abordagens complementares, sendo a lipase Candida
antarctica B o biocatalisador mais eficiente para as modificações seletivas de ambos, o tianfenicol
e o correspondente diacetato do tianfenicol, respectivamente. Os melhores resultados foram
obtidos nas reações de acilação com diferentes ésteres vinílicos, contendo cadeias carbônicas de
comprimento variável, levando aos correspondentes 3’-monoésteres com excelentes rendimentos e
em tempos reacionais curtos.As condições reacionais foram analisadas em termos de concentração
de substrato, carga de enzima e tipo de doador de acila. Foi demonstrado o reuso da enzima por cinco
vezes sem perda significativa de atividade. Alternativamente, a abordagem hidrolítica propiciou a
preparação de alguns 1’-monoésteres em bons rendimentos, ainda que os níveis de reatividade e
seletividade foram mais baixos que os obtidos na reação complementar de acetilação.
The lipase-catalyzed synthesis of thiamphenicol derivatives has been studied through
complementary acylation and hydrolytic approaches, finding Candida antarctica lipase B as the
most efficient biocatalyst for the selective modification of both thiamphenicol and thiamphenicol
diacetate, respectively. The best results have been obtained using acylation reactions with different
vinyl esters of variable length, yielding the corresponding 3’-monoesters with excellent yields and
in short reaction times. The conditions have been analyzed in terms of substrate concentration,
enzyme loading and type of acyl donor. The reuse of the enzyme for five-times without significant
loss of the activity has also been demonstrated. Alternatively, the hydrolytic approach has allowed
the preparation of some 1’-monoesters in good yields, although the reactivity and selectivity levels
were lower than the ones achieved for the complementary acetylation reaction.
Keywords: biocatalysis, lipases, medium engineering, regioselective processes, thiamphenicol
Introduction
displaying a similar spectrum of therapeutic activities
(Figure 1). Also a fluorinated derivative of thiamphenicol
(florfenicol, 3) is currently indicated for the treatment of
bovine respiratory disease, among others.
Thetotalasymmetricsynthesisof(1R,2R)-thiamphenicol
has received attention in recent years, considering different
Thiamphenicol (1), also known as thiophenicol,
dextrosulphenidol or 2,2-dichloro-N-{(1R,2R)-1,3-
dihydroxy-1-[4-(methylsulfonyl)phenyl]propan-2-yl}
acetamide, is a potent antibiotic used especially for
veterinary applications against infectious diseases, and
has also been successfully tested in humans.1 It possesses
remarkable activity against both Gram negative and Gram
positive microorganisms. Structurally, thiamphenicol
is the methylsulfonyl analogue of chloramphenicol (2),
Figure 1. Chemical structures of thiamphenicol (1), chloramphenicol
(2) and florfenicol (3).
*e-mail: mcdmatto@ufc.br, vgs@uniovi.es

988
Regioselective Preparation of Thiamphenicol Esters Through Lipase-Catalyzed Processes
J. Braz. Chem. Soc.
key steps for the introduction of chirality, such as Sharpless
asymmetric epoxidation of an allylic alcohol intermediate,2
Ag(I) promoted asymmetric bromo-methoxylation of an
α,β-unsaturated amide precursor,3 chemical resolution of
a racemic propanediol,4 or the addition of an enantiopure
sulfinimine to the lithium enolate of methyl bromoacetate.5
Enzymes have received considerable attention in recent
years, as they display high selectivities under environmentally
and mild reaction conditions.6 For instance, adequate
thiamphenicol precursors have been obtained by means of
efficient stereoselective methods using lipases, amidases
and hydroxynitrile lyases, starting from an adequate amide,
ester or aldehyde precursor, respectively.7 Nowadays,
thiamphenicol is a commercially available product, and a
challenging task is the development of selective chemical
modifications for the synthesis of thiamphenicol analogs
with improved therapeutical activities.
The solubility is a key issue for multiple in vivo
applications, and thiamphenicol presents a negligible
solubility in water. On the other hand, organic solvents
such as diethyl ether, ethyl acetate or chloroform allow
its complete solubility at high substrate concentrations.
Preparation of thiamphenicol derivatives remains currently
as an attractive area, because of the possible improved
biological activities shown by them in comparison with the
parent substrate. To solve this problem, biocatalysis appears
as a useful tool allowing the development of regioselective
processes. Thus, different enzymes, especially hydrolases,
have allowed the selective modification of identical
functional groups in different regions of a target molecule.8
Lipases are hydrolytic enzymes widely used for
synthetic transformations.9 Advantages arise from
their complementary action in hydrolytic and synthetic
procedures, especially for transesterification reactions.
Their versatility to act in both media offers a myriad of
opportunities, especially when there is a clear interest
to obtain monoesters starting from diols or diesters.
This fact clearly improves traditional chemical methods,
which usually require tedious sequences of protection and
deprotection steps on selected functionalities.
in good yields by means of acylation procedures and
complementary hydrolytic reactions.
Experimental
General methods
Chemical reagents were purchased from different
commercial sources and used without further purification.
Candida antarctica lipase type B (CAL-B, Novozyme 435,
7300 PLU g-1) and Rhizomucor miehei lipase (Lipozyme
RM IM, < 15% in weight) were a gift from Novozymes
(Denmark). AK lipase from Pseudomonas fluorescens
(22100 U g-1), and Candida rugosa lipase (965 U mg-1 of
solid using olive oil) were purchased from Sigma-Aldrich.
Solvents were distilled over an adequate desiccant under
nitrogen. Flash chromatographies were performed using
silica gel 60 (230-240 mesh). IR spectra were recorded
on using NaCl plates or KBr pellets in a Perkin-Elmer
1720-X F7.
High performance liquid chromatography (HPLC)
analyses were carried out in a Hewlett Packard 1100
chromatograph UV detector at 210 nm using a Daicel
CHIRALCEL IA or a CHIRALPAK AS column
(25 cm × 4.6 mm i.d.). For the measurement of the
conversion values, calibration curves were done for all
the compounds involved in the enzymatic processes.
Standard solutions at 1000 ppm substrate concentration
were prepared and from these ones the 50, 100, 150,
200 and 250 ppm solutions were prepared. Analyses of
individual substrates were done in the HPLC conditions
indicated for each organic compound. Absorbance values
were obtained and used with the Origin® program to attain
graphics and equations for calibration curves. Correlation
coefficients were in all cases r2 > 0.999 for curve fitting.
Reaction times were as follows: (i) reactions for the
synthesis of acetylated derivatives: Column Chiralpak
AS with a 0.8 mL min-1 flow using 25% 2-propanol/
hexane as eluent at 40 ºC: 1 (12.3 min), 5a (22.7 min),
7a (27.5 min) and 8a (13.4 min); (ii) reactions for the
synthesis of propionated derivatives: Column Chiralpak
IA with a 0.8 mL min-1 flow using 25% 2-propanol/hexane
as eluent at 40 ºC: 1 (7.9 min), 5b (10.1 min), 7b (9.5 min)
and 8b (8.8 min); (iii) reactions with vinyl decanoate:
Column Chiralpak AS with a 0.8 mL min-1 flow using
25% 2-propanol/hexane as eluent at 40 ºC: 1 (12.3 min),
5c (6.5 min) and 7c (10.6 min); (iv) reactions with vinyl
laurate: Column Chiralpak AS with a 0.8 mL min-1 flow
using 25% 2-propanol/hexane as eluent at 40 ºC: 1
(12.3 min), 5d (5.9 min) and 7d (9.9 min); (v) reactions
with vinyl palmitate: Column Chiralpak AS with a
In fact, the regioselective formation of a thiamphenicol
ester from 1 was successfully demonstrated for the first time
by Carrea and co-workers, obtaining in almost quantitative
conversion the 3’-palmitate derivative by lipase G-catalyzed
acylation reaction in acetone.10 More recently, Lin and
co-workers reported the synthesis of thiamphenicol vinyl
esters employing the Mucor miehei lipase and divinyl esters
of variable length.11 Herein, we wish to report an effective
and selective general methodology for the production of
thiamphenicol esters, the action of lipases being optimized
for the isolation of the corresponding target compounds

Vol. 25, No. 6, 2014
Silva et al.
989
0.8 mL min-1 flow using 20% 2-propanol/hexane as eluent
at 40 ºC: 1 (12.3 min), 5e (5.6 min) and 7e (11.1 min).
General procedure for the acylation reaction of
(–)-thiamphenicol with vinyl decanoate (4c). Enzyme
recycling studies
General procedure for the enzymatic acylation reaction of
(–)-thiamphenicol with vinyl esters 4a-e
To a suspension of (–)-thiamphenicol (1, 50 mg,
0.14 mmol) and CAL-B (50 mg) in dry MeCN (1.4 mL),
vinyl decanoate (4c, 182 µL, 5 eq, 0.71 mmol) was added,
and the reaction shaken at 20 ºC and 250 rpm under nitrogen
atmosphere.Aliquots were regularly analyzed by HPLC and
the reaction stopped when complete consumption of the
starting material was observed after 1 h. Finally, the enzyme
was filtered off and washed with EtOAc (3 × 10 mL), the
solvent evaporated under reduced pressure, and the reaction
crude purified by flash chromatography on silica gel (60%
EtOAc/hexane) affording the corresponding 3’-monoester
5c (96% isolated yield). This experiment was repeated with
the recycled enzyme for five times without any loss of the
enzyme activity.
To a suspension of (–)-thiamphenicol (1, 50 mg,
0.14 mmol) and CAL-B (50 mg) in dry MeCN (1.4 mL),
the corresponding vinyl ester 4a-e (5 eq, 0.70 mmol)
was added under nitrogen atmosphere, and the reaction
shaken at 20 ºC and 250 rpm. Aliquots were regularly
analyzed by HPLC and the reaction stopped when
complete consumption of the starting material was
observed (1-3 h, see Table 1). Finally, the enzyme was
filtered off and washed with EtOAc (3 × 5 mL), the solvent
evaporated under reduced pressure, and the reaction crude
purified by flash chromatography on silica gel (60%
EtOAc/hexane), affording the corresponding monoesters
5a (96%), 5b (98%), 5c (94%), 5d (96%) and 5e (98%).
General procedure for the chemical synthesis of diesters
7a-e from (–)-thiamphenicol (1)
Table 1. Regioselective lipase-catalyzed acylation of thiamphenicol (1,
150 mmol L-1) using CAL-B and vinyl esters 4a-e (5 eq) for the production
of 3’-monoesters 5a-e at 20 ºC and 250 rpm in acetonitrile
4-(N,N-dimethylamino)pyridine (DMAP, 3.4 mg,
0.028 mmol), Et3N (58 µL, 0.421 mmol) and the
corresponding anhydride 6a,b or acid chloride 6c-e
(0.421 mmol) were added to a thiamphenicol (1, 100 mg,
0.281 mmol) solution in dry CH2Cl2 (2.8 mL) under
nitrogen atmosphere. The reaction was stirred at room
temperature during 1 h and after that time the solvent was
evaporated in a rotary evaporator. The resulting crude
was purified by flash chromatography on silica gel (30%
EtOAc/hexane) to afford the desired diester 7a (37%),
7b (54%), 7c (61%), 7d (50%) and 7e (41%).
entry
R
time / h
5a-e / %a
> 99 (96)
> 99 (98)
> 99 (94)
> 99 (96)
> 99 (98)
1
2
3
4
5
CH3− (a)
1
1
3
3
3
CH3CH2− (b)
CH3(CH2)8− (c)
CH3(CH2)10− (d)
CH3(CH2)14− (e)
General procedure for the chemical acylation reaction of
(–)-thiamphenicol (1) to obtain monoesters 5a-e and 8a-e
for analytical purposes
aConversion values measured by HPLC. Isolated yields after purification
on silica gel chromatography appear in parentheses.
DMAP (11 mg, 0.09 mmol), Et3N (204 µL, 1.47 mmol)
and the corresponding anhydride 6a,b or acid chloride 6c-e
(1.47 mmol) were added to a thiamphenicol (1, 347 mg,
0.98 mmol) solution in dry CH2Cl2 (4.0 mL) under nitrogen
atmosphere. The reaction was stirred at room temperature
during 1 h and after that time the solvent was evaporated
in a rotary evaporator. The resulting crude was purified by
flash chromatography on silica gel (30% EtOAc/hexane)
to afford the standards of the desired monoesters 5a-e and
8a-e.
General procedure for the enzymatic acylation reaction of
(–)-thiamphenicol with vinyl propionate (4b) at 1 g scale
To a suspension of (–)-thiamphenicol (1, 1 g, 2.74 mmol)
and CAL-B (1 g) in dry MeCN (28 mL), vinyl propionate
(4b, 298 µL, 2.74 mmol) was added under nitrogen
atmosphere, and the reaction shaken at 20 ºC and 250 rpm.
Aliquots were regularly analyzed by HPLC and the reaction
stopped when complete consumption of the starting
material was observed after 2 h. Finally, the enzyme was
filtered off and washed with EtOAc (3 × 10 mL), the
solvent evaporated under reduced pressure, and the reaction
crude purified by flash chromatography on silica gel (60%
EtOAc/hexane), affording the corresponding monoester 5b
(97% isolated yield).
General procedure for the enzymatic hydrolysis reaction of
(–)-thiamphenicol diesters 7a,b
A suspension of diester 7a,b (30 mg) and CAL-B or
Candida rugosa lipase (CRL) (ratio 1:1 m/m with respect to
the diester) in a mixture of phosphate buffer 100 mmol L-1

990
Regioselective Preparation of Thiamphenicol Esters Through Lipase-Catalyzed Processes
J. Braz. Chem. Soc.
pH 7.0/MeCN (80/20 v/v), was shaken at 20 ºC and
250 rpm. The reaction was stopped after 24-48 hours, and
the enzyme filtered off. The organic solvent was evaporated
and the residue extracted with EtOAc (3 × 10 mL). Finally,
the solvent was evaporated under reduced pressure, and the
reaction crude purified by flash chromatography on silica
gel (60% EtOAc/hexane).
(t, 3H, J 6.6 Hz), 1.26 (s, 16H), 1.63 (t, 2H, J 7.1 Hz),
2.35 (t, 2H, J 7.6 Hz), 3.00 (s, 3H), 4.17-4.44 (m, 3H),
5.04 (s, 1H), 5.78 (s, 1H), 6.97 (d, 1H, J 8.7 Hz), 7.52 (d,
2H, J 8.2 Hz), 7.77 (d, 2H, J 8.2 Hz); 13C NMR (CDCl3,
75.5 MHz) d 14.2, 22.8, 25.0, 29.3-29.8, 32.1, 34.3, 44.7,
54.4, 62.9, 66.3, 70.8, 127.1, 127.5, 139.8, 147.0, 164.5,
174.3; HRMS (ESI+) calcd. for C24H37Cl2NNaO6S (M):
560.1639; found: 560.1611; [α]D20 = +0.62 (c 1.0, CHCl3).
Full characterization of novel compounds 5a-e, 7a-e, and
8a-b
(2’R,3’R)-Thiamphenicol-3’-palmitate (5e): Rf (60%
EtOAc/hexane): 0.54; m.p. 108-110 ºC; IR (NaCl)
(2’R,3’R)-Thiamphenicol-3’-acetate (5a): Rf (60%
EtOAc/hexane): 0.18; m.p. 133-135 ºC; IR (NaCl)
max/cm-1: 3055, 2987, 1743, 1699, 1521, 1421, 1316, 1265,
n
max/cm-1: 3019, 2927, 2855, 2346, 1733, 1698, 1521, 1420,
1316, 1265, 1151 cm-1; 1H NMR (CDCl3, 300.13 MHz) d
0.87 (t, 3H, J 6.7 Hz), 1.25 (s, 24H), 1.62 (t, 2H, J 7.0 Hz),
2.35 (t, 2H, J 7.6 Hz), 3.00 (s, 3H), 4.17-4.44 (m, 3H),
5.03 (s, 1H), 5.77 (s, 1H), 6.96 (d, 1H, J 8.7 Hz), 7.51 (d,
2H, J 8.2 Hz), 7.76 (d, 2H, J 8.2 Hz); 13C NMR (CDCl3,
75.5 MHz) d 14.3, 22.8, 25.0, 29.3-29.6, 32.1, 34.3, 44.7,
54.4, 62.9, 66.3, 70.9, 127.1, 127.5, 139.8, 147.0, 164.4,
174.4; HRMS (ESI+) calcd. for C28H45Cl2NNaO6S (M):
616.2247; found: 616.2237; [α]D20 = +0.38 (c 1.0, CHCl3).
n
1151 cm-1; 1H NMR (CDCl3, 300.13 MHz) d 2.08 (s, 3H),
3.00 (s, 3H), 4.17-4.40 (m, 3H), 5.04 (s, 1H), 5.80 (s, 1H),
7.03 (d, 1H, J 8.5 Hz), 7.50 (d, 2H, J 8.2 Hz), 7.74 (d, 2H
J 8.2 Hz); 13C NMR (CDCl3, 75.5 MHz) d 20.9, 44.6, 54.3,
63.1, 66.3, 70.7, 127.1, 127.4, 139.6, 147.2, 164.5, 171.4;
HRMS (ESI+) calcd. for C14H17Cl2NNaO6S (M): 420.0046;
found: 420.0057; [α]D20 = +0.75 (c 0.8, CHCl3).
(2’R,3’R)-Thiamphenicol-3’-propionate (5b): Rf (60%
EtOAc/hexane): 0.33; m.p. 111-113 ºC; IR (NaCl) nmax/cm-1:
3055, 2987, 2305, 1743, 1699, 1521, 1421, 1315, 1265,
(2’R,3’R)-Thiamphenicol-1’,3’-diacetate (7a): Rf
(60% EtOAc/hexane): 0.26; m.p. 115-117 ºC; IR (NaCl)
n
max/cm-1: 3055, 2987, 2412, 2305, 1750, 1703, 1523,
1
1
1151, 1088 cm-1; H NMR (CDCl3, 300.13 MHz) d 1.13
1421, 1316, 1265, 1153, 1049 cm-1; H NMR (CDCl3,
300.13 MHz) d 2.07 (s, 3H), 2.16 (s, 3H), 3.05 (s, 3H), 4.02
(dd, 1H, J 6.0, 12.0 Hz), 4.16 (dd, 1H, J 6.0, 12.0 Hz), 4.57-
4.66 (m, 1H), 5.88 (s, 1H), 6.05 (d, 1H, J 6.0 Hz), 6.89 (d,
1H, J 9.3 Hz), 7.55 (d, 2H, J 8.2 Hz), 7.93 (d, 2H, J 8.2 Hz);
13C NMR (CDCl3, 75.5 MHz) d 20.8, 20.9, 44.5, 52.9, 62.3,
66.2, 73.1, 127.9, 128.1, 141.1, 142.6, 164.4, 169.8, 170.7;
HRMS (ESI+) calcd. for C16H19Cl2NNaO7S (M): 462.0151;
found: 462.0176; [α]D20 = −3.89 (c 1.0, CHCl3).
(t, 3H, J 7.5 Hz), 2.36 (q, 2H, J 7.5 Hz), 3.00 (s, 3H), 4.18-
4.23 (m, 1H), 4.29-4.41 (m, 2H), 5.04 (s, 1H), 5.80 (s, 1H),
7.27 (d, 1H, J 8.5 Hz), 7.50 (d, 2H, J 8.2 Hz), 7.74 (d, 2H,
J 8.2 Hz); 13C NMR (CDCl3, 75.5 MHz) d 9.1, 27.6, 44.6,
54.4, 63.0, 66.6, 70.8, 127.1, 127.4, 139.6, 147.2, 164.5,
174.8; HRMS (ESI+) calcd. for C15H19Cl2NNaO6S (M):
434.0202; found: 434.0218; [α]D20 = +0.11 (c 1.0, CHCl3).
(2’R,3’R)-Thiamphenicol-3’-decanoate (5c): Rf (60%
EtOAc/hexane): 0.67; m.p. 85-87 ºC; IR (NaCl) nmax/cm-1:
3055, 2928, 2856, 2305, 1743, 1699, 1519, 1421, 1316,
(2’R,3’R)-Thiamphenicol-1’,3’-dipropionate (7b): Rf
(60% EtOAc/hexane): 0.46; m.p. 105-107 ºC; IR (NaCl)
1265, 1151 cm-1; H NMR (CDCl3, 300.13 MHz) d 0.88
n
max/cm-1: 3059, 2928, 2305, 1743, 1707, 1527, 1463, 1313,
1
(t, 3H, J 7.1 Hz), 1.26 (s, 12H), 1.63 (t, 2H, J 7.2 Hz), 2.36
(t, 2H, J 7.4 Hz), 3.02 (s, 3H), 4.16-4.47 (m, 3H), 5.04 (s,
1H), 5.30 (s, 2H), 5.70 (s, 1H), 6.92 (d, 1H, J 8.8 Hz), 7.54
(d, 2H, J 8.2 Hz), 7.82 (d, 2H, J 8.2 Hz); 13C NMR (CDCl3,
75.5 MHz) d 14.3, 22.8, 25.0, 29.3-29.5, 32.0, 34.3, 44.7,
54.4, 62.7, 66.3, 70.9, 127.1, 127.6, 140.1, 146.7, 164.5,
174.4; HRMS (ESI+) calcd. for C22H33Cl2NNaO6S (M):
532.1307; found: 532.1298; [α]D20 = −0.54 (c 0.8, CHCl3).
1267, 1151, 1084 cm-1; 1H NMR (CDCl3, 300.13 MHz) d
1.12 (t, 3H, J 7.5 Hz), 1.14 (t, 3H, J 7.5 Hz), 2.34 (q, 2H,
J 7.5 Hz), 2.43 (q, 2H, J 7.5 Hz), 3.04 (s, 3H), 4.02 (dd, 1H,
J 6.0, 12.0 Hz), 4.17 (dd, 1H, J 6.0, 12.0 Hz), 4.60 (m, 1H),
5.88 (s, 1H), 6.04 (d, 1H, J 6.0 Hz), 6.88 (d, 1H, J 9.3 Hz),
13
7.54 (d, 2H, J 7.5 Hz), 7.93 (d, 2H, J 7.5 Hz); C NMR
(CDCl3, 75.5 MHz) d 9.0, 9.1, 27.4, 27.6, 44.6, 53.0, 62.3,
66.2, 72.8, 127.9, 128.1, 141.1, 142.7, 164.3, 173.3, 174.1;
HRMS (ESI+) calcd. for C18H23Cl2NNaO7S (M): 490.0464;
found: 490.0484; [α]D20 = −3.7 (c 1.0, CHCl3).
(2’R,3’R)-Thiamphenicol-3’-laurate (5d): Rf (60%
EtOAc/hexane): 0.47; m.p. 94-96 ºC; IR (NaCl) nmax/cm-1:
3055, 2928, 2855, 2306, 1733, 1699, 1519, 1421, 1316,
(2’R,3’R)-Thiamphenicol-1’,3’-didecanoate (7c): Rf
(60% EtOAc/hexane): 0.87; m.p. 82-84 ºC; IR (NaCl)
1
1265, 1151 cm-1; H NMR (CDCl3, 300.13 MHz) d 0.88

Vol. 25, No. 6, 2014
Silva et al.
991
n
max/cm-1: 3054, 2928, 2856, 2305, 1743, 1703, 1522, 1421,
J 8.3 Hz); 13C NMR (CDCl3, 75.5 MHz) d 21.1, 44.6, 55.6,
61.0, 66.4, 73.4, 128.0, 128.2, 140.9, 143.4, 164.7, 170.4;
HRMS (ESI+) calcd. for C14H17Cl2NNaO6S (M): 420.0083;
found: 420.0046; [α]D20 = −3.6 (c 0.5, CHCl3).
1317, 1265, 1153 cm-1; H NMR (CDCl3, 300.13 MHz)
d 0.88 (t, 6H, J 6.8 Hz), 1.26 (br s, 24H), 1.63 (t, 4H,
J 4.6 Hz), 2.35 (t, 2H, J 7.7 Hz), 2.42 (t, 2H, J 7.7 Hz),
3.05 (s, 3H), 4.02 (dd, 1H, J 6.0, 12.0 Hz), 4.16 (dd, 1H,
J 6.0, 12.0 Hz), 4.55-4.62 (m, 1H), 5.30 (s, 2H), 5.86
(s, 1H), 6.05 (d, 1H, J 6.0 Hz), 6.76 (d, 1H, J 9.3 Hz),
1
(2’R,3’R)-Thiamphenicol-1’-propionate (8b): Rf (60%
EtOAc/hexane): 0.25; m.p. 109-111 ºC; IR (NaCl)
n
max/cm-1: 3497, 3289, 2929, 1727, 1684, 1556, 1462, 1369,
13
7.55 (d, 2H, J 8.2 Hz), 7.95 (d, 2H, J 8.2 Hz); C NMR
(CDCl3, 75.5 MHz) d 14.3, 22.8, 24.9, 25.0, 29.3-29.6,
32.0, 34.2, 34.3, 44.6, 53.1, 62.2, 66.3, 72.8, 127.9 , 128.2,
141.3, 142.7, 164.6, 172.7, 173.5; HRMS (ESI+) calcd.
for C32H51Cl2NNaO7S (M): 686.2664; found: 686.2656;
[α]D20 = −1.24 (c 1.0, CHCl3).
1300, 1151, 1084 cm-1; 1H NMR (CDCl3, 300.13 MHz) d
1.14 (t, 3H, J 7.5 Hz), 2.42 (q, 2H, J 7.5 Hz), 3.05 (s, 3H),
3.49 (dd, 1H, J 6.0, 12.0 Hz), 3.66 (dd, 1H, J 6.0, 12.0 Hz),
4.31-4.38 (m, 1H), 5.93 (s, 1H), 6.19 (d, 1H, J 7.2 Hz),
7.09 (d, 1H, J 9.2 Hz), 7.63 (d, 2H, J 8.3 Hz), 7.93 (d, 2H,
J 8.3 Hz); 13C NMR (CDCl3, 75.5 MHz) d 9.1, 27.8, 44.6,
55.7, 61.0, 66.5, 73.3, 128.0, 128.2, 140.9, 143.6, 164.6,
173.8; HRMS (ESI+) calcd. for C15H19Cl2NNaO6S (M):
434.0202; found: 434.0241; [α]D20 = −4.4 (c 1.0, CHCl3).
(2’R,3’R)-Thiamphenicol-1’,3’-dilaurate (7d): Rf (60%
EtOAc/hexane): 0.75; m.p. 48-50 ºC; IR (NaCl) nmax/cm-1:
3055, 2928, 2855, 2306, 1743, 1703, 1522, 1421, 1317,
1265, 1152 cm-1; 1H NMR (CDCl3, 300.13 MHz) d 0.88 (t,
6H, J 6.6 Hz), 1.25 (br s, 32H), 1.61 (s, 4H), 2.32 (t, 2H,
J 8.6 Hz), 2.40 (t, 2H, J 8.6 Hz), 3.04 (s, 3H), 4.02 (dd, 1H,
J 6.0, 12.0 Hz), 4.19 (dd, 1H, J 6.0, 12.0 Hz), 4.55-4.64
(m, 1H), 5.86 (s, 1H), 6.04 (d, 1H, J 6.0 Hz), 6.76 (d, 1H,
J 9.3 Hz), 7.55 (d, 2H, J 8.2 Hz), 7.95 (d, 2H, J 8.2 Hz);
13C NMR (CDCl3, 75.5 MHz) d 14.2, 22.8, 24.9 , 29.2-
29.6, 32.0, 34.2, 44.6, 53.1, 62.2, 66.2, 72.8, 127.9, 128.1,
141.2, 142.8, 164.2, 172.7, 173.5; HRMS (ESI+) calcd.
for C36H59Cl2NNaO7S (M): 742.3294; found: 742.3282;
[α]D20 = −0.64 (c 1.0, CHCl3).
Results and Discussion
Regioselective acylation of thiamphenicol using vinyl esters
Based on the excellent results achieved in the selective
acylation of chloramphenicol (2),12 reactions were carried
out in the presence of 5 eq of different vinyl esters
presenting different chain length such as acetate (4a),
propionate (4b), decanoate (4c), laurate (4d) and palmitate
(4e). Candida antarctica lipase type B (CAL-B, ratio 1:1
m/m with respect to 1) was selected as biocatalyst, using
acetonitrile as solvent (Table 1).
All acylation reactions occurred with complete
regioselectivity towards the primary alcohol, obtaining
monoesters 5a-e with full conversion after 24 h. Then,
we decided to monitor the reactions at shorter reaction
times (1-3 h). Satisfyingly, the biotransformations
reached complete conversions, requiring slightly longer
reaction times for those vinyl esters with a longer chain
(4c-e, entries 3-5). The corresponding thiamphenicol
monoesters 5a-e were isolated with excellent yields after
flash chromatography purification. These results are highly
interesting as the chemical acylation of thiamphenicol
with anhydrides or chloride acids led preferentially to the
diacylated products 7a-e (37-61%) in the presence of an
excess of the acyl donor, while only a minor amount of the
3’-monoester derivatives were obtained using 1.5 eq of the
corresponding acylating agents (Scheme 1).
(2’R,3’R)-Thiamphenicol-1’,3’-dipalmitate (7e): Rf (60%
EtOAc/hexane): 0.86; m.p. 47-49 ºC; IR (NaCl) nmax/cm-1:
3054, 2927, 2854, 2306, 1743, 1703, 1522, 1466, 1317,
1265, 1153 cm-1; 1H NMR (CDCl3, 300.13 MHz) d 0.87 (t,
6H, J 6.7 Hz), 1.24 (br s, 48H), 1.61 (s, 4H), 2.32 (t, 2H,
J 8.6 Hz), 2.42 (t, 2H, J 8.6 Hz), 3.04 (s, 3H), 4.02 (dd, 1H,
J 6.0, 12.0 Hz), 4.19 (dd, 1H, J 6.0, 12.0 Hz), 4.55-4.64
(m, 1H), 5.86 (s, 1H), 6.04 (d, 1H, J 6.0 Hz), 6.83 (d, 1H,
J 9.3 Hz), 7.55 (d, 2H, J 8.2 Hz), 7.93 (d, 2H, J 8.2 Hz);
13C NMR (CDCl3, 75.5 MHz) d 14.2, 22.8, 24.9, 29.2-
29.6, 32.0, 34.2, 44.6, 53.1, 62.2, 66.2, 72.8, 127.9, 128.1,
141.2, 142.8, 164.2, 172.7, 173.5; HRMS (ESI+) calcd.
for C44H75Cl2NNaO7S (M): 854.4555; found: 854.4534;
[α]D20 = −1.6 (c 1.0, CHCl3).
(2’R,3’R)-Thiamphenicol-1’-acetate (8a): Rf (60%
EtOAc/hexane): 0.15; IR (NaCl) nmax/cm-1: 3328, 3019,
2928, 1735, 1688, 1533, 1410, 1303, 1225, 1147 cm-1;
1H NMR (CDCl3, 300.13 MHz) d 2.13 (s, 3H), 3.06 (s, 3H),
3.49 (dd, 1H, J 6.0, 12.0 Hz), 3.66 (dd, 1H, J6.0, 12.0 Hz),
4.33-4.38 (m, 1H), 5.94 (s, 1H), 6.18 (d, 1H, J 7.2 Hz),
7.10 (d, 1H, J 9.2 Hz), 7.63 (d, 2H, J 8.3 Hz), 7.93 (d, 2H,
At this point, different experiments were carried out
in order to find optimum conditions for the production of
the corresponding monoesters. Focusing in the economic
assessment of the biotransformation, the enzyme loading
was reduced five times, recovering ester 5a after 2 h and

992
Regioselective Preparation of Thiamphenicol Esters Through Lipase-Catalyzed Processes
J. Braz. Chem. Soc.
(6a) in the presence of triethylamine (Et3N) and 4-(N,N-
dimethylamino)pyridine (DMAP) in dichloromethane
(Scheme 1). Then, a set of hydrolases were employed for
the development of the hydrolytic reactions over diester
7a. Hence, CAL-B, Candida antarctica lipase type A
(CAL-A), Candida rugosa lipase (CRL), lipase AK from
Pseudomonas fluorescens (AK), Rhizomucor miehei
lipase (RML), Thermomyces lanuginosus lipase (TLL)
and CLEA-alcalase from Bacillus subtilis esterase were
tried (Table 2).
Scheme 1. Chemical synthesis of thiamphenicol diester derivatives 7a-e.
After enzymatic screening, hydrolytic activity was
only observed with CAL-B and CRL, which hydrolyzed
preferentially the ester at the 3’-position at 20 ºC when the
reactions were carried out in a system composed of MeCN
and phosphate buffer pH 7.0 (entries 1 and 2). The best
results were obtained with CAL-B, which afforded 8a in
a good yield after 24 h at 20 ºC (81%, entry 1), finding the
diol as a minor product (4%). As previously mentioned,
due to the complementarity of lipases, the same reaction
center reacted in comparison with the CAL-B catalyzed
acetylation of thiamphenicol. Longer reaction times
favored the formation of diol 1. On the other hand, CRL
also showed activity yielding 33% of 8a, although most of
the diacetate remained unreacted (64%) after 24 h (entry
2). Unfortunately, longer reaction times (48 h) led to a
dramatic loss in the selectivity of the process favoring the
formation of the diol at prolonged reaction times (entry
3). A great improvement was achieved when doubling the
CRL loading, yielding a 61% of 8a (entry 4).
In order to improve the selectivity of this enzyme
towards the formation of 1’-monoester in the hydrolysis
of 7a, other cosolvents such as 1,4-dioxane, ethanol,
isopropanol (IPA), isoamyl alcohol (IAA), tetrahydrofuran
and 2-methyltetrahydrofuran were considered, but in all
cases lower activities and selectivities were observed
(entries 5-10). A similar trend was witnessed when using
higher temperatures in acetonitrile (entries 11 and 12).
Finally, we decided to expand this approach to another
diester containing a longer chain such as the dipropionate
derivative 7b, but a decrease in the selectivity was observed
in the reaction catalyzed by CAL-B, obtaining a complex
mixture of thiamphenicol (4%), monoester 8b (69%),
monoester 5b (18%) and diester 7b (9%). Based on
previous results with the chloramphenicol derivatives,12
diesters 7c-e with longer acyl chains were not studied due
to the lack of CAL-B reactivity.
45 min with complete conversion and 98% isolated yield.
Reactions were also performed with a higher thiamphenicol
concentration (250 mmol L-1), finding for vinyl propionate
(4b) the formation of the final product 5b in quantitative
conversion and excellent isolated yield (97%) in a similar
reaction time.
Scaling-up protocols of these acylation reactions
were performed using 0.5 and 1 g of the starting material
at 250 mmol L-1 substrate concentration. The processes
were developed at 20 ºC using 5 eq of vinyl acetate (4a),
obtaining the 3’-monoester 5a with total conversion after 2
h. Remarkably, the reactions occurred also with complete
conversion employing only 2 eq of 4a but requiring in this
case 3 h. Similarly, the reaction with 1 eq of vinyl propionate
(4b) allowed the formation of the monoester 5b in 97%
isolated yield at 100 mmol L-1 thiamphenicol concentration at
20 ºC. Finally, the recycling of the biocatalyst was analyzed,
taking as model reactions the synthesis of monoesters 5a
and 5c, which were studied through acylation reactions of
thiamphenicol using 5 eq of vinyl acetate (4a) and vinyl
decanoate (4c), respectively. Having in mind the reactivity
of both esters, the biotransformations were carried out for
1 h with 4a, and 3 h for 4c, at 20 ºC and 100 mmol L-1
substrate concentration. It was demonstrated that CAL-B
can be reused up to five times keeping unaltered its activity
and regioselectivity in the presence of vinyl esters with both
short and long chain esters.
Regioselective hydrolysis of thiamphenicol esters
Lipase-catalyzed reactions offer clear advantages in
terms of selectivity but also due to the complementarity
of hydrolytic and synthetic reactions. For this reason,
hydrolytic transformations over thiamphenicol diesters
were studied in order to provide a useful route towards the
1’-monoesters. These compounds are difficult to achieve
by conventional chemical methods due to the lack of
selectivity but also because of the hindered accessibility
towards this 1’-position. Diacetylated compound 7a
was prepared using a small excess of acetic anhydride
Conclusions
In summary, an efficient lipase-catalyzed strategy
has been developed for the selective 3’-acylation of

Vol. 25, No. 6, 2014
Silva et al.
993
Table 2. Regioselective lipase-catalyzed hydrolysis of diester 7a using an organic cosolvent and phosphate buffer pH 7.0 (20:80) at 250 rpm
Monoacetate Diacetate 7a Monoacetate
entry
Enzyme
Substrate:Enzymea
Solvent
Temp. / ºC
time / h
Diol 1 / %b
5a / %b
/ %b
15
64
24
29
73
87
83
82
82
82
41
12
8a / %b
1
CAL-B
CRL
CRL
CRL
CRL
CRL
CRL
CRL
CRL
CRL
CRL
CRL
1:1
1:1
1:1
1:2
1:1
1:1
1:1
1:1
1:1
1:1
1:1
1:1
MeCN
MeCN
MeCN
MeCN
1,4-Dioxane
EtOH
20
20
20
20
20
20
20
20
20
20
30
40
24
24
48
24
24
24
24
24
24
24
24
24
4
1
0
2
81
2
33
3
13
4
35
6
28
4
61
5
3
7
17
6
2
5
6
7
IPA
1
4
12
8
IAA
1
3
14
9
THF
1
5
12
10
11
12
2-Me-THF
MeCN
MeCN
0
3
15
6
22
34
31
30
24
am/m ratio; bpercentage of compounds determined by HPLC. CAL-B: Candida antarctica lipase type B; CRL: Candida rugosa lipase; IPA: isopropanol;
IAA: isoamyl alcohol.
thiamphenicol with different vinyl esters of variable
length, obtaining the final derivatives in excellent isolated
yields (> 93%). Candida antarctica lipase B allows the
formation of monoacylated derivatives in short reaction
times with complete selectivities. The scaling of these
processes has being optimized for 1 g scale. Hydrolytic
experiments have been attempted in order to obtain the
complementary monoacylated esters at 1’-position. In these
experiments only CAL-B and CRL have shown remarkable
activity, finding CAL-B as a more suitable enzyme for the
production of the 1’-monoacetylated derivative, while the
use of the dipropionated thiamphenicol derivative led to a
decrease in the selectivity of the process. This is another
example where the use of enzymes for regioselective
modifications can overcome the limitations of traditional
synthetic methods for the selective transformation of
polyhydroxylated compounds.
Acknowledgments
We thank Novozymes for the generous gift of CAL-B
(Novozyme 435) and Rhizomucor miehei lipase. The
authors thank the Brazilian and Spanish agencies CAPES-
DGU (Process: 149/07), CNPq, FUNCAP, PRONEX,
Ministerio de Ciencia e Innovación (MICINN, Projects
CTQ2007-61126 and CTQ2011-24237) for fellowships and
financial support. I. L. (Ramón y Cajal program) thanks
MICINN for a personal grant.
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Submitted: January 24, 2014
Published online: April 8, 2014