Biotechnological properties of sponges from northeast Brazil: Cliona varians as a Biocatalyst for Enantioselective Reduction of Carbonyl Compounds

Article abstract of DOI:10.21577/0103-5053.20180165

To research the potential ability of whole marine sponges to act as biocatalysts, this paper describes for the first time the employment of whole Cliona varians sponge in the stereoselective reduction of prochiral α-keto esters and isatin to the corresponding chiral alcohols. The addition of D-fructose, D-glucose or sucrose remarkably increased the conversion ratios and stereoselectivities by this marine sponge. Furthermore, in the presence of D-glucose and D-maltose, the reduction of isatin by C. varians afforded the corresponding 3-hydroxyindolin-2-one with high conversions (85-90percent) and good enantioselectivities (60-74percent). These results showed that the marine sponge presents great potential to be used as biocatalyst for stereoselective reduction of carbonyl compounds.

Full text of DOI:10.21577/0103-5053.20180165

http://dx.doi.org/10.21577/0103-5053.20180165
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-9, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Biotechnological Properties of Sponges from Northeast Brazil: Cliona varians as a
Biocatalyst for Enantioselective Reduction of Carbonyl Compounds
Valéria B. Riatto,*,a,b,c Mauricio M. Victor,a,b,c Jaqueline F. Sousaa,b,c and Carla Menegolad
aDepartamento de Química Orgânica, Instituto de Química,
Universidade Federal da Bahia, 40170-115 Salvador-BA, Brazil
bInstituto Nacional de Ciência e Tecnologia (INCT) em Energia e Ambiente,
Universidade Federal da Bahia, 40170-115 Salvador-BA, Brazil
cCentro Interdisciplinar de Energia e Ambiente (CIENAM),
Universidade Federal da Bahia, 40170-115 Salvador-BA, Brazil
dCentro de Estudos Costeiros, Limnológicos e Marinhos (CECLIMAR),
Universidade Federal do Rio Grande do Sul, Campus Litoral Norte, 95625-000 Imbé-RS, Brazil
To research the potential ability of whole marine sponges to act as biocatalysts, this paper
describes for the first time the employment of whole Cliona varians sponge in the stereoselective
reduction of prochiral α-keto esters and isatin to the corresponding chiral alcohols. The addition of
D-fructose, D-glucose or sucrose remarkably increased the conversion ratios and stereoselectivities
by this marine sponge. Furthermore, in the presence of D-glucose and D-maltose, the reduction of
isatin by C. varians afforded the corresponding 3-hydroxyindolin-2-one with high conversions (85-
90%) and good enantioselectivities (60-74%). These results showed that the marine sponge presents
great potential to be used as biocatalyst for stereoselective reduction of carbonyl compounds.
Keywords: α-keto-esters, isatin, biotransformations, marine sponge, Cliona varians
Introduction
In recent years, there are several reports about
the possibility of using parts of fresh plants tissue as
biocatalysts, since different oxidoreductases and the
cofactor regeneration system are present in the plant
cell.5 Moreover, application of comminuted tissue of ripe
vegetable roots in biotransformations, instead of isolated
enzymes, is possible due to the group of enzymes excreted
to extracellular medium that are able to accept xenobiotic
substrates.6
Several vegetable species from our Brazilian
biodiversity were investigated as biocatalysts in different
organic reactions, such as: cushcush (Dioscorea trifida
L.) and cactus (Opuntia ficus-indica (L.) Miller),7 manioc
(Manihot esculenta),8 passion fruit (Passiflora edulis)9 and
sugar cane (Saccharum officinarum).10 However, Brazilian
marine biodiversity remains practically unexplored in the
search for new biocatalysts systems.
Biotransformation of exogenous substrates have been
widely studied and used to prepare chiral compounds. The
use of enzymes in catalytic reactions, such as asymmetric
reduction of prochiral ketones, is one of the most important
and practical reaction for producing chiral alcohols,
which can be transformed into various functionalities,
to synthesize industrially important chemicals such as
pharmaceuticals and other commercial products.1,2
The utilization of biocatalytic systems have got benefits
of economically viable, ecologically favorable and more
sustainable than current chemical technologies, due to their
inherent advantages of higher selectivity, milder conditions
and comparatively cheaper resources.3 However, a limiting
factor of use of enzymes is their specificity towards the
substrate.4 Therefore, searching for new enzymes from
easily available natural sources is an important task in the
field of asymmetric organic synthesis.
Sponges (phylum Porifera) are the most primitive
multicellular animals, which have existed for
700-800 million years, demonstrating relatively little
differentiation and tissue coordination.11 To date, more
*e-mail: vriatto@ufba.br

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Biotechnological Properties of Sponges from Northeast Brazil
J. Braz. Chem. Soc.
than 18,000 species of sponges were described, of which
approximately 8,800 are considered valid, most occurring
in marine environments, with about 1% being found in
freshwater habitats,12 but as many as twice that number
are thought to exist. They are sessile invertebrates with a
wide variety of colors, shapes and consistencies that have
developed efficient defense mechanisms against predators.
Marine sponges are filter feeding porous animals and
usually harbor a remarkable array of microorganisms
in their mesohyl tissues as transient and resident
endosymbionts. The marine sponge-microbial interactions
are highly complex and, in some cases, the relationships
are thought to be truly symbiotic or mutualistic rather than
temporary associations resulting from sponge filter-feeding
activity. The marine sponge-associated microorganisms are
a fascinating source for various biomolecules that are of
potential interest to several biotechnological industries.13
Cliona varians (Dushassaing & Michelotti, 1864)
is a species of encrusting and excavating demosponge
belonging to the family Clionaidae. It is found in shallow
water in the Caribbean Sea and its range extends from Cuba
and Mexico south toVenezuela and Brazil. It usually grows
on massive corals, covering its surface, but is sometimes
found on some other calcareous substrate such as mollusk
shells, crustacean carapaces or limestone rocks. It can form
thick encrustations that may cover several square meters
of substratum. Oscules are relatively large (10 to 30 mm
wide), paler and slightly elevated, surrounded by projecting
rims or small raised papillae 2 to 5 mm wide. The color is
usually dull orange to tan or brownish.14
Literature reports that marine-derived organisms are
sources of diverse enzymes suitable for biotransformation
or biocatalytic reactions of organic compounds.15 In
addition, several marine sponge-derived fungi have been
applied in the bioreduction of iodoacetophenones,16
azido-ketones17 and isatin.18 However, to date, there is
no report about the use of marine sponge’s tissue in
biotransformation reactions.
Concerning to the biotechnological properties of
Cliona spp., methods of sponge cell culture for the
production of biologically active metabolites are nowadays
developed using primmorphs, three-dimensional aggregates
comprising proliferating and differentiating sponge cells,
which can be maintained for long periods. The primmorphs
have been generated from a wide range of sponges,
including Cliona celata Grant, 1826.19 In the coming years,
it should become clear whether primmorphs can be scaled
up sufficiently to overcome the supply problem for many
promising drug leads.20
report an investigation of the whole marine sponge C. varians
as reducing agent of α-keto esters and isatin (1H-indole-2,3-
dione) (1) to the corresponding chiral alcohols.
Experimental
Isatin 1, ethyl pyruvate (2), α-ketoglutaric acid (6),
D-maltose, D-fructose, L-aspartate and NaBH4 were
purchased from Aldrich Chemical Co., Milwaukee,
WI, USA. D-Glucose and glycine were purchased from
Merck, Darmstadt, Germany. L-Alanine and L-glutamate
were purchased from Vetec, São Paulo, Brazil. All
reagents were used without further purification. Alcohols
were distilled immediately prior to use. The reactions
involving anhydrous solvents were carried out under argon
atmosphere. Column chromatography was performed using
silica gel (70-230 Mesh from Acros) and reactions were
monitored by thin-layer chromatography (TLC) Silica TLC
plates (Macherey-Nagel, Germany).
1H nuclear magnetic resonance (NMR) spectra were
recorded in CDCl3 solution at 500 or 200 MHz and
13C NMR spectra in CDCl3 solution at 125 or 50 MHz
on a Varian equipment mod. Inova 500 and on a Bruker
equipment mod. DPX 200. Chemical shifts, given on the
d scale, were referenced to the residual, undeuterated
portion using of the deuterated CDCl3 solvent (dH 7.27) in
relation to tetramethylsilane (TMS). Infrared (IR) spectra
were recorded on a Shimadzu FTIR spectrophotometer
mod. IR Affinity-1S. Optical rotations were measured at
25 °C in a PerkinElmer 343 Polarimeter at 589 nm (sodium
D line). The conversions and enantiomeric excess (ee)
of α-hydroxy esters 2a-5a and 3-hydroxyindolin-
2-one (1a) were determined by gas chromatography (GC)
analysis using an Agilent Technologies equipment model
7820A, with flame ionization detector (FID), employing
Agilent Cyclosil-B Capillary Column 30% heptakis-
(2,3-di-O-methyl-6-O-t-butyldimethylsilyl)-β-cyclodextrin
(30 m × 0.25 mm × 0.25 mm) as stationary phases. Nitrogen
was used as the carrier gas, flow rate of 1.18 mL min-1
in split mode; the injector temperature and detector
temperature were set at 220 °C; the column temperature
was programmed at 80 °C for 10 min, 2 °C min-1 from 80
to 200 °C for 10 min.
Biocatalysts
Specimens of the marine sponge Cliona varians were
collected in tide pools (0-0.5 m depth) at Praia do Forte
Beach, Mata de São João (12°33’50”S/37°59’36”W) at
coastline of Bahia (Northeastern Brazil). The species
C. varians was identified by marine biologist Dr Carla
As a part of our ongoing project in the application of
novel biocatalysts from the Brazilian biodiversity,7 herein we

Vol. 00, No. 00, 2018
Riatto et al.
3
Menegola. Voucher specimen (No. 4039, 4040 and 4032)
has been deposited at the Zoology Museum of the Biology
Department, Federal University of Bahia, Salvador, Brazil.
The collect was authorized by Environmental Minister
(permanent license SISBIO-ICMBio-MMA No. 11793-3).
After the collection, the fresh sponges were transported in
a thermal box at 4 °C, and then washed exhaustively with
sea water and frozen at –18 °C until use. All sponges were
collected on sunny days, when ocean water temperature
was between 25 and 27 °C.
Synthesis of standard racemic dimethyl 2-hydroxyglutarate
(3a)21
To a solution of dimethyl 2-oxoglutarate (3) (100 mg,
0.57 mmol) in MeOH (4.0 mL) at 0 °C, under argon
atmosphere, it was added NaBH4 (1.5 eq., 0.86 mmol).
The mixture was stirred 24 h at room temperature and
citric acid was added until the pH reaches 5-6. Then,
silica gel (150 mg) was added to de reaction mixture and
the solvent was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography
(EtOAc:hexane 1:1) to afford dimethyl 2-hydroxyglutarate
(3a) (79.2 mg, 0.45 mmol) as a colorless oil, in 78% yield;
1H NMR (CDCl3, 500 MHz) d 1.94 (dtd, J 14.3, 8.1 and
6.2 Hz, 1H), 2.15 (dddd, J 14.3, 8.1, 7.3, 4.2 Hz, 1H), 2.44
(ddd, J 16.6, 8.1, 6.2 Hz, 1H), 2.53 (ddd, J 16.6, 8.1, 7.3 Hz,
1H), 2.96 (br s, 1H), 3.69 (s, 3H), 3.80 (s, 3H), 4.25 (dd,
J 8.1, 4.2 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d 29.2,
29.5, 51.8, 52.7, 69.5, 173.6, 175.1; IR (film) n / cm-1 3483,
2954, 1739, 1438, 1261, 1107; GC (FID) tR (S): 35.3 min
and tR (R): 35.8 min; TLC (EtOAc:hexane 1:1) Rf = 0.30.
Synthesis of α-keto esters
To a solution of α-ketoglutaric acid (6) (3.3 mmol,
500 mg) in freshly distilled dry alcohol (methanol, ethanol
or isopropanol) (10 mL) at room temperature, it was
added p-toluenesulfonic acid (PTSA) (0.4 mmol, 65 mg).
The mixture was refluxed 24 h and sequentially NaHCO3
(0.8 mmol, 70 mg) was added. After stirring 30 min at
room temperature, the solvent was removed in vacuo and
the crude mixture was diluted with ethyl acetate (10 mL),
dried over MgSO4 and concentrated under reduced pressure.
Silica gel column chromatography (50% EtOAc in hexanes,
v/v) of the crude product afforded the respective ester as
a colorless oil.
General procedure for the preparation of standard
racemic diethyl 2-hydroxyglutarate (4a) and diisopropyl
2-hydroxyglutarate (5a)21
To a solution of 2-oxoglutarate ester (0.6 mmol) in
tetrahydrofuran (THF) (20.0 mL) at 0 °C, under argon
atmosphere was added NaBH4 (2.0 eq., 1.2 mmol). The
mixture was stirred 24 h at room temperature and citric
acid was added until the pH reaches 5-6. Then, silica
gel (250 mg) was added to the reaction mixture and the
solvent was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography
(EtOAc:hexane 1:1) to afford the respective α-hydroxy
esters as a colorless oil.
Dimethyl 2-oxoglutarate (3)
1
Yield 78%; H NMR (CDCl3, 500 MHz) d 2.69 (t,
J 5.9 Hz, 2H), 3.17 (t, J 5.9 Hz, 2H), 3.69 (s, 3H), 3.89
(s, 3H); 13C NMR (CDCl3, 125 MHz) d 27.4, 34.2, 51.9,
53.0, 160.9, 172.4, 192.2; IR (film) n / cm-1 3005, 2958,
1782, 1732, 1438, 1168, 1083; GC (FID) tR: 39.3 min; TLC
(EtOAc:hexane 1:1) Rf = 0.40.
Diethyl 2-oxoglutarate (4)
1
Yield 76%; H NMR (CDCl3, 200 MHz) d 1.22 (t,
J 7.0 Hz, 3H), 1.34 (t, J 7.0 Hz, 3H), 2.63 (t, J 6.5 Hz,
2H), 3.12 (t, J 6.5 Hz, 2H), 4.10 (q, J 7.0 Hz, 2H), 4.29
(q, J 7.0 Hz, 2H); 13C NMR (CDCl3, 50 MHz) d 13.9,
14.1, 27.7, 34.1, 60.8, 62.5, 160.5, 171.9, 192.7; IR (film)
n / cm-1 2983, 1737, 1255, 1082; GC (FID) tR: 46.9 min;
TLC (EtOAc:hexane 1:1) Rf = 0.60.
Diethyl 2-hydroxyglutarate (4a)
1
Yield 33%; H NMR (CDCl3, 200 MHz) d 1.23 (t,
J 7.7 Hz, 3H), 1.27 (t, J 7.7 Hz, 3H), 1.84-1.98 (m, 1H),
2.06-2.22 (m, 1H), 2.41-2.49 (m, 2H), 3.10 (br s, 1H),
4.05-4.27 (m, 5H); 13C NMR (CDCl3, 50 MHz) d 14.1, 29.3,
29.7, 60.5, 61.8, 69.5, 173.1, 174.6; IR (film) n / cm-1 3500,
2954, 1732, 1440, 1215, 1107; GC (FID) tR (R): 41.3 min
and tR (S): 41.8 min; TLC (EtOAc:hexane 1:1) Rf = 0.40.
Diisopropyl 2-oxoglutarate (5)
1
Yield 70%; H NMR (CDCl3, 200 MHz) d 1.23 (d,
J 6.2 Hz, 6H), 1.35 (d, J 6.2 Hz, 6H), 2.63 (t, J 5.4 Hz,
2H), 3.13 (t, J 5.4 Hz, 2H), 5.00 (hept., J 6.2 Hz, 1H),
5.15 (hept., J 6.2 Hz, 1H); 13C NMR (CDCl3, 50 MHz) d
21.6, 21.8, 28.0, 34.2, 68.3, 70.8, 160.2, 171.5, 193.1; IR
(film) n / cm-1 2983, 1730, 1469, 1377, 1215, 1078, 758;
GC (FID) tR: 41.5 min; TLC (EtOAc:hexane 1:1) Rf = 0.70.
Diisopropyl 2-hydroxyglutarate (5a)
1
Yield 54%; H NMR (CDCl3, 200 MHz) d 1.21 (d,
J 6.3 Hz, 6H), 1.26 (d, J 6.3 Hz, 6H), 1.79-1.97 (m, 1H),
2.05-2.21 (m, 1H), 2.37-2.51 (m, 2H), 3.00 (br s, 1H),
4.16 (dd, J 3.9, 7.7 Hz, 1H), 4.93-5.14 (m, 2H); 13C NMR
(CDCl3, 50 MHz) d 21.8, 29.4, 30.1, 67.8, 69.6, 69.7, 172.7,

4
Biotechnological Properties of Sponges from Northeast Brazil
J. Braz. Chem. Soc.
174.2; IR (film) n / cm-1 3483, 2954, 1739, 1438, 1281,
1107; GC (FID) tR (R): 37.6 min and tR (S): 37.9 min; TLC
(EtOAc:hexane 1:1): Rf = 0.50.
pieces of sponges (40 g) in aqueous 0.85% (v/v) sea salt
solution (120 mL). The mixtures were incubated in a shaker
(180 rpm) at 25 ºC for 3 days, when sugars were used as
additives, and 5 days when amino acids were used. The
reaction process was monitored by TLC. Each individual
suspension was filtered, and the residue was washed with
water. The aqueous solutions were then extracted with
EtOAc (3 × 30 mL), and the organic soluble fractions were
dried with MgSO4 and evaporated under reduced pressure.
The residues were filtered on a short silica gel column,
using CHCl3 as eluent, to afford the reduced products.
Conversions and enantiomeric excess were quantified
employing chiral GC analyses. The stereochemistry of the
major enantiomer was determined by measuring the specific
rotation of the mixture obtained after the biotransformation
step and comparing it with the specific rotation value
reported in literature.
Synthesis of standard racemic 3-hydroxyindolin-2-one (1a)22
To a solution of NaBH4 (3 mmol, 1.5 eq., 112.4 mg)
in 12 mL of 1:1 CH2Cl2:EtOH, at 0 °C, under argon
atmosphere, was added a solution of commercially available
isatin 1 (2.0 mmol, 294 mg) in 8 mL of 1:1 CH2Cl2:EtOH.
The mixture was stirred during 5 min at 0 °C and then
2.0 mL of water was added. Sequentially, the mixture was
stirred 10 min at room temperature and it was extracted with
CH2Cl2 (3 × 10 mL). The organic phase was washed with
brine (10 mL), dried over MgSO4, filtered and concentrated.
The crude product was purified by recrystallization
(EtOAc/hexane) to afford 3-hydroxyindolin-2-one (1a)
(194 mg, 1.3 mmol) in 65% yield, as a beige solid; 1H NMR
(DMSO-d6, 500 MHz) d 3.14 (br s, 1H), 4.81 (s, 1H), 6.78
(d, J 7.8 Hz, 1H), 6.95 (t, J 7.5 Hz, 1H), 7.20 (t, J 7.8 Hz,
1H), 7.27 (d, J 7.5 Hz, 1H), 10.21 (s, 1H); 13C NMR
(DMSO-d6, 125 MHz) d 69.6, 109.9, 121.9, 125.2, 129.3,
129.7, 142.6, 178.3; IR (film) n / cm-1 3379, 2926, 2341,
1728, 1618; GC (FID) the column did not separate the
enantiomers, tR: 71.4 min; GC (FID) isatin: tR: 73.3 min;
TLC (EtOAc:hexane 1:1) Rf = 0.20.
(–)-(S)-Dimethyl 2-hydroxyglutarate (3a)
L-Aspartate (additive): 65% conversion; 16% ee (S);
25
25
[α]D –1.0 (c 0.38, MeOH); Lit. (S): [α]D –5.2 (c 0.38,
MeOH).23
(+)-(R)-Dimethyl 2-hydroxyglutarate (3a)
Sucrose (additive): 70% conversion; 28% ee (R);
25
[α]D +1.5 (c 0.38, MeOH). D-Fructose (additive): 46%
Synthesis of standard racemic ethyl 2-hydroxypropanoate
(2a)21
conversion; 12% ee (R); [α]D25 +1.0 (c 0.38, MeOH).
To a solution of commercially available ethyl pyruvate (2)
(100 mg, 0.87 mmol) in EtOAc (4.0 mL) at 0 °C, under
argon atmosphere, was added NaBH4 (0.5 eq., 16.7 mg,
0.44 mmol). The mixture was stirred 24 h at room
temperature and was added acetic acid until the pH reaches
4-5. Then, the solvent was evaporated under reduced
pressure to afford ethyl 2-hydroxypropanoate (2a) (44.9 mg,
0.38 mmol) as a colorless oil in 46% yield; IR (film)
n / cm-1 3441, 2985, 2939, 1732, 1215, 1134, 428; GC
(FID) tR (S): 28.8 min and tR (R): 29.3 min; GC (FID) ethyl
pyruvate tR: 30.2 min; TLC (EtOAc:hexane 1:1) Rf = 0.35.
(–)-(S)-Diethyl 2-hydroxyglutarate (4a)
Sucrose (additive): 20% conversion; 24% ee (S); 11%
yield; [α]D25 –1.0 (c 0.45, MeOH). L-Aspartate (additive):
40% conversion; 38% ee (S); 26% yield; [α]D25 –1.5 (c 0.45,
25
MeOH); Lit. (S): [α]D −4.1 (c 0.45, MeOH).23
(+)-(R)-Diethyl 2-hydroxyglutarate (4a)
D-Fructose (additive): 35% conversion; 60% ee (R);
18% yield; [α]D25 +2.2 (c 0.45, MeOH).
(–)-(S)-Diisopropyl 2-hydroxyglutarate (5a)
Sucrose (additive): 81% conversion; 47% ee (S); 30%
yield; [α]D25 –1.6 (c 0.88, MeOH). D-Fructose (additive):
68% conversion; 75% ee (S); 45% yield; [α]D25 –2.5 (c 0.88,
MeOH); Lit. (S): [α]D25 –3.5 (c 0.88, MeOH).23
Bioreduction of substrates
Bioreduction reactions were performed using various
additives (e.g., sugars such as sucrose, D-glucose,
D-maltose, and D-fructose or amino acids such as
L-alanine, glycine, L-glutamate, and L-aspartate).
The specimens were rinsed with aqueous 0.85% (v/v)
sea salt solution and cut into small pieces (approx. 1 cm
long slice) with a sterile knife. In separate experiments,
substrates 1-5 (1.15 mmol) and additive (38.0 mmol)
were individually added to a suspension of the freshly
(R)-3-Hydroxyindolin-2-one (1a)
D-Fructose (additive): 90% conversion; 44% ee (R);
42% yield; [α]D25 +3.1 (c 1.00, MeOH); Lit. (R): [α]D25 +7.0
(c 1.00; MeOH).24
(–)-(S)-3-Hydroxyindolin-2-one (1a)
Absence of additive: 23% conversion; 71% ee (S); 15%

Vol. 00, No. 00, 2018
Riatto et al.
5
yield; [α]D25 –5.0 (c 1.00, MeOH). D-Glucose (additive):
85% conversion; 74% ee (S); 46% yield; [α]D25 –5.2 (c 1.00,
MeOH). D-Maltose (additive): 90% convrsion; 60% ee (S);
45% yield; [α]D25 –4.2 (c 1.00, MeOH).
Synthesis of substrates
Isatin 1 and ethyl pyruvate (2) employed were
commercially available. Dimethyl 2-oxoglutarate (3),
diethyl 2-oxoglutarate (4), diisopropyl 2-oxoglutarate (5)
were obtained in 78, 76 and 70% yields, respectively,
from the esterification of α-ketoglutaric acid (6) with
the corresponding alcohol, in the presence of PTSA
(Scheme 1).
(–)-(S)-2-Hydroxypropanoate (2a)
L-Glutamate (additive): 19% conversion; 70% ee; 10%
yield; [α]D25 –10.4 (c 2.0, CHCl3). L-Aspartate (additive):
20% conversion; 70% ee; 10% yield; [α]D25 –10.2 (c 2.0,
CHCl3). Sucrose (additive): 48% conversion; 84% ee; 37%
25
yield; [α]D –12.5 (c 2.0, CHCl3). D-Glucose (additive):
Bioreductions of isatin 1 and α-keto esters 2-5 by marine
sponge C. varians
58% conversion; 72% ee; 49% yield; [α]D25 –10.6 (c 2.0,
CHCl3). D-Fructose (additive): 74% conversion; 54% ee;
50% yield; [α]D25 –8.0 (c 2.0, CHCl3). Lit. (S): [α]D25 –15
(c 2.0, CHCl3).25
Stereoselectivity of microbe-catalyzed reduction of
keto esters can be controlled by the addition of additives
in the reaction systems.30 Furthermore, the conversion
ratios of α-keto esters to the corresponding α-hydroxy
esters, using biotransformation, were increased by addition
of sugars or amino acids into the reaction mixture as an
additive.31 In recent years, the effect of additives on the
reduction of α-keto esters, using marine actinomycetes,
were investigated. Among various additives (e.g., sugars
or amino acids), the introduction of L-glutamate or
sucrose remarkably increased the conversion ratio of the
reduction.32
With this goal in mind, dimethyl 2-oxoglutarate (3)
was chosen as a model substrate to explore the effect of
additives on conversion and stereoselectivity of sponge
reduction. The first series of bioreductions was carried
out at 25 °C and the effect of additives on the reduction
of 3, using marine sponge C. varians, was investigated.
Experiments were performed in triplicate and the results
are presented in Table 1.
Results and Discussion
Choose of substrates
Enantiomerically pure α-hydroxy esters are very
important chiral building blocks for the synthesis
of a variety of natural products and biologically
active molecules.26 Although there are several reports
available in the literature dealing with stereoselective
reduction of ketones and β-keto esters mediated by
biocatalysis, the bioreduction of α-keto esters is less
common. Chiral 3-hydroxyindolin-2-one (1a) is a
product of the enantioselective reduction of isatin 1
and showed pharmacological potential to antiallergic,
anti-inflammatory and anticancer activities.27 However,
few studies in the literature have focused on the
biotransformation of isatin derivatives by whole cells.28
The structure of the substrate is a determining factor
for the stereochemical course of bioreduction reactions.29
The choice of structures was made in order to verify the
influence of the relative steric volumes of the alkoxy
groups of 2-oxoglutarate, compared with isatin 1 and ethyl
pyruvate (2). The α-keto esters choices as substrate were
dimethyl-2-oxoglutarate (3), diethyl-2-oxoglutarate (4) and
diisopropyl 2-oxoglutarate (5).
It was found that C. varians did not reveal a capacity
for the bioreduction of 3 in the absence of additive
(Table 1, entry 1), whereas no reaction was observed in
the presence of L-alanine and glycine (Table 1, entries 2
and 3). L-Aspartate, sucrose and D-fructose showed better
conversion to α-hydroxy ester 3a, but low selectivities
(12-28%) (Table 1, entries 4, 6 and 8). L-Glutamate,
D-glucose and D-maltose showed low conversions to 3a
Scheme 1. Synthesis of α-keto esters 3-5.

6
Biotechnological Properties of Sponges from Northeast Brazil
J. Braz. Chem. Soc.
Table 1. Bioreduction reactions of dimethyl 2-oxoglutarate (3) by C. varians
entry
Additive
none
Conversiona / %
eea / %
1
2
3
4
5
6
7
8
9
−
−
L-alanine
glycine
−
−
−
−
L-aspartate
L-glutamate
sucrose
65
10
70
20
46
20
16 (S)-3a
42 (S)-3a
28 (R)-3a
8 (R)-3a
12 (R)-3a
20 (S)-3a
D-glucose
D-fructose
D-maltose
aConversion and enantiomeric excess (ee) were determined by chiral GC.
(Table 1, entries 5, 7 and 9). Depending on the additive
employed, different enantiomerically enriched α-hydroxy
esters 3a were obtained. The addition of the additives
L-aspartate, L-glutamate and D-maltose produce the
S enantiomer (Table 1, entries 4, 5 and 9), while the addition
of the additives sucrose, D-glucose and D-fructose produce
the R isomer (Table 1, entries 6, 7 and 8). Therefore, their
structures are not predictable via the empirical Prelog rule,33
which describes the stereoselectivity of the attack of the
hydride ion to the carbonyl group. The configuration was
dependent on the nature of the additive employed.
seems that the increase in the concentration of reduced
nicotinamide-adenine dinucleotide (NADH or NADPH)
due to the oxidative degradation of the additive by the
enzymes present in the marine sponge would accelerate
the stereoselective reduction of α-keto esters to the
corresponding optically pure alcohols.31
In order to expand the study to other α-keto esters,
it was performed a series of bioreductions employing
diethyl 2-oxoglutarate 4 and diisopropyl 2-oxoglutarate
5 as substrates. L-Aspartate, sucrose and D-fructose were
employed as additives for these studies. The obtained
results are described in Table 2.
The mechanism underlying the increase in the
conversion due to the addition of sugars or amino acids
as additives to the reaction mixtures is not clear. It
The products obtained through the use of L-aspartate
and sucrose showed predominance of the S configuration.
Table 2. Bioreduction reactions of α-keto esters 4 and 5 by C. varians
entry
R
Additive
sucrose
Conversiona / %
eea / %
24 (S)-4a
60 (R)-4a
38 (S)-4a
47 (S)-5a
75 (S)-5a
−
1
2
3
4
5
6
Et
Et
Et
iPr
iPr
iPr
25
35
40
81
70
−
D-fructose
L-aspartate
sucrose
D-fructose
L-aspartate
aConversion and enantiomeric excess (ee) were determined by chiral GC.

Vol. 00, No. 00, 2018
Riatto et al.
7
Table 3. Bioreduction reactions of ethyl pyruvate (2) by C. varians
Only D-fructose was able to reduce the α-keto ester 4 to
corresponding alcohol 4a with R-configuration (Table 2,
entry 2). Unfortunately, the reduction of diethyl α-keto
ester 4 by C. varians, in the presence of sucrose, D-fructose
and L-aspartate gave the corresponding α-hydroxy ester 4a
with low conversions (25-40%) (Table 2, entries 1-3) and
moderate ee (60% R-configuration). It is interesting to note
that no reduction of diisopropyl α-keto ester 5 in presence
of L-aspartate (Table 2, entry 6) was observed, while
sucrose and D-fructose gave the corresponding α-hydroxy
ester 5a with good conversions (70-81%) and moderate to
good selectivities (ee 47-75%, Table 2, entries 4 and 5). The
(−)-(S)-diisopropyl 2-hydroxyglutarate (5a), produced by
bioreduction in the presence of D-fructose, was isolated
by column chromatography with 45% yield and 75% ee.
After these encouraging results, we decided to extend
our studies toward the analysis of isatin 1 and ethyl pyruvate
(2), in order to investigate the substrate specificity of marine
sponge C. varians. The biorreduction reactions of ethyl
pyruvate (2) were carried out at 25 °C, the experiments
were performed in triplicate and the results are presented
in Table 3.
The use of L-aspartate, L-glutamate, sucrose, D-glucose
and D-fructose additives led to the formation of 2a with
predominance of the S-configuration. No reaction was
observed in the presence of L-alanine, glycine and in the
absence of additive (Table 3, entries 1-3). However, the low
to moderate conversions did not improve by the addition of
some additives such as L-glutamate, L-aspartate, sucrose
and D-glucose (19-58%) (Table 3, entries 4-7). Whereas,
the reduction of ethyl pyruvate (2) by C. varians in presence
of D-fructose gave the corresponding alcohol 2a with 78%
entry
Additive
none
Conversiona / %
eea / %
−
1
2
3
4
5
6
7
8
0
L-alanine
glycine
0
−
0
−
L-glutamate
L-aspartate
sucrose
19
20
48
58
74
70 (S)-2a
70 (S)-2a
84 (S)-2a
72 (S)-2a
54 (S)-2a
D-glucose
D-fructose
aConversion and enantiomeric excess (ee) were determined by chiral GC.
conversion. On the other hand, the introduction of sucrose
improved the stereoselectivity of the α-hydroxy ester
(S)-2a (84% ee) (Table 3, entry 6), moreover the effects of
other additives (L-glutamate, L-aspartate and D-glucose)
gave goods stereoselectivities of the produced alcohol
(70-72% ee) (Table 3, entries 4, 5 and 7). The (−)-(S)-ethyl
lactate (2a), produced in the presence of sucrose, was isolated
by column chromatography with 37% yield and 84% ee.
The biorreduction reactions of isatin 1 were done at
25 °C. The experiments were performed in triplicate and
the results are presented in Table 4.
The products obtained through the use of sucrose,
D-glucose and D-maltose showed predominance of the
Table 4. Bioreduction reactions of isatin 1 by C. varians
entry
Additive
none
Conversiona / %
eeb / %
71 (S)-1a
−
1
2
3
4
5
6
7
8
23
−
L-alanine
L-glutamate
L-aspartate
sucrose
−
−
−
−
5
61 (S)-1a
74 (S)-1a
44 (R)-1a
60 (S)-1a
D-glucose
D-fructose
D-maltose
85
90
90
aConversions were determined by GC; benantiomeric excess (ee) were determined by specific rotation.

8
Biotechnological Properties of Sponges from Northeast Brazil
J. Braz. Chem. Soc.
S-configuration. Only D-fructose was able to reduce
the isatin 1 to corresponding (R)-(3)-hydroxyindolin-
2-one (1a), with 90% conversion and 44% ee (Table 4,
entry 7). As can be seen in Table 4, entry 1, the marine
sponge had natural activity for reducing isatin 1, and
showed good stereoselectivity (71% ee) in the absence
of additive. In comparison with the reactions of α-keto
esters 2-3, the behavior was different because they were
not reduced in the absence of additives. Furthermore,
in the presence of sugars D-glucose and D-maltose,
the reduction of isatin 1 afforded the corresponding
(−)-(S)-(3)-hydroxyindolin-2-one 1a with high conversions
(85-90%) and good enantioselectivities (60-74% ee). In
the literature, whole cells of marine-derived fungi were
used to reduce isatin 1 to (−)-(S)-1a.18 The best conversion
was obtained by fungi Westerdykella sp. CBMAI 1679
(89%), but with only 18% ee, while best enantiomeric
excess was obtained by fungi Aspergillus sydowii
CBMAI 935 (66% ee), but with only 9% conversion. In
our approach, the (−)-(S)-3-hydroxyindolin-2-one (1a),
produced by marine sponge in the presence of D-glucose,
was isolated by column chromatography with 46% yield
and 74% ee.
Supplementary Information
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
Acknowledgments
The authors are grateful to Brazilian Agencies CNPq
(National Council for Scientific and Technological
Development), FAPESB (Baiana Foundation for the
Support of Scientific and Technological Development), and
INCT E&A (National Institute for Science and Technology
for Energy and Environment) for financial support. The
authors thank LABAREMN (UFBA) for conducting NMR
spectra and Prof Gustavo Henrique Ribeiro Viana (UFSJ)
for the support to synthesis of keto esters and NMR spectra.
We are grateful to all the researchers and students who
contributed collecting specimens: RogerioVianna (FURG),
Tiago dos Anjos dos Santos, Daniele Menezes dos Santos,
Myrla Josefa Santana Rocha and Danilo Pereira Pinto
(UFBA). C. M. received grants and fellowship of FAPESB
and CNPq (Project ESCUBRA-Sponges from the segment
Cuba-Brasil).
Conclusions
References
The results described in this paper demonstrate that the
C. varians, a species of locally available marine sponge
studied, has enzymatic systems with the required skill
to reduce prochiral α-keto esters 2-5 and isatin 1 to the
corresponding chiral alcohols. As a result of this study,
it is clear that an unexpected opportunity has arisen to
be able to establish new applications for marine sponges,
especially for those species which do not have any other
reported practical utility. The encouraging results obtained
here using whole marine sponge for biocatalysis may
offer new strategies for the reduction of selected prochiral
α-keto esters and α-keto lactam as a critical step in the
synthetic organic pathway, specifically avoiding the use
of costly and non-sustainable metal reducing agents and
organic solvents that are commonly utilized in organic
synthesis. In conclusion, four chiral α-hydroxy esters
and 3-hydroxyindolin-2-one (1a) were synthesized with
satisfactory conversions (70-81%) and medium to good
enantiomeric excess (42-84% ee), thus revealing the marine
sponge C. varians to be a promising biocatalyst for the
production of key intermediates. Further investigations are
currently ongoing to try to expand the specificity and to
explore novel catalytic activities of this new biocatalytic
agent.
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Submitted: May 15, 2018
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Published online: August 27, 2018
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