Esterase screening using whole cells of Brazilian soil microorganisms

Article abstract of DOI:10.1590/S0103-50532010000800011

A miniaturized enzymatic assay using fluorescent probes to reveal esterase producing microorganisms was optimized and applied to screen 64 soil bacterial strains. The best results were validated using traditional non-fluorogenic assays with acetyl and propanoyl phenylethanol to confirm the miniaturized results. The most active microorganisms belong to the genus Bacillus showing esterase activity and good enantiomeric ratios for the resolution of phenylethanol derivatives (E > 30). Part of the microorganisms are kept in our laboratory in glycerol or freezedried and the best microorganisms will be deposited in the CBMAI/CPQBA/Unicamp culture collection.

Full text of DOI:10.1590/S0103-50532010000800011

J. Braz. Chem. Soc., Vol. 21, No. 8, 1484-1489, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Esterase Screening using Whole Cells of Brazilian Soil Microorganisms
Simone M. Mantovani, Luciana G. de Oliveira and Anita J. Marsaioli*
Instituto de Química, Universidade Estadual de Campinas, CP 6154,
13083-970 Campinas-SP, Brazil
Um ensaio enzimático miniaturizado com sondas fluorescentes para triagem rápida de
microrganismos produtores de esterases foi implementado e aplicado a 64 linhagens de bactérias
de solo. Os melhores resultados foram validados por métodos convencionais e substratos não
fluorogênicos (feniletanol acetilado e propanoilado) e confirmaram os resultados obtidos nas
triagens rápidas. Os microrganismos que apresentaram atividades enzimáticas mais relevantes
(razão enantiomérica E > 30) na hidrólise de ésteres e na obtenção de boas razões enantioméricas
foram identificados como pertencentes ao gênero Bacillus. Parte dos microrganismos se encontra
preservada em glicerol e/ou liofilizada no IQ/Unicamp e três linhagens serão depositadas na
CBMAI do CPQBA/Unicamp como depósitos abertos.
A miniaturized enzymatic assay using fluorescent probes to reveal esterase producing
microorganisms was optimized and applied to screen 64 soil bacterial strains. The best results
were validated using traditional non-fluorogenic assays with acetyl and propanoyl phenylethanol
to confirm the miniaturized results. The most active microorganisms belong to the genus Bacillus
showing esterase activity and good enantiomeric ratios for the resolution of phenylethanol
derivatives (E > 30). Part of the microorganisms are kept in our laboratory in glycerol or freeze-
dried and the best microorganisms will be deposited in the CBMAI/CPQBA/Unicamp culture
collection.
Keywords: soil bacteria, miniaturized screening with fluorescent probes, esterases
Introduction
These enzymes accept a wide range of substrates and
have been used to catalyze hydrolysis, esterification and
transesterification reactions that make these biocatalysts
versatile and attractive tools in industry.7,8 Other important
points to be considered are co-factor independence and high
stability in non-aqueous media.9,10
The most important feature distinguishing lipases and
esterases is substrate specificity. Lipases preferentially
promote the hydrolysis of water-insoluble esters such as
triglycerides composed of long chain fatty acids while
esterases prefer short-chain acid triglycerides. Another
distinction based on protein structure shows that most
lipases possess a lid covering the active site, a feature
that is absent in esterases. Moreover, most lipases
present an interfacial activation dependence on substrate
concentration.6
The search for novel biocatalysts involves exploring
nature and the environment to find new active enzymes. The
immense Brazilian biodiversity especially from soil, that is
a rich source of microorganisms, has motivated the search
for new biocatalysts with special characteristics aiming at
biotechnological applications.1 These microorganisms can
metabolize and biotransform regio- and enantioselectively
an enormous range of natural and synthetic organic
compounds using different enzymes.2 Among these, the
hydrolases (E.C. 3) are the most useful biocatalysts in
chemistry with outstanding applications in food chemistry,
detergents, treatment of oily residues and in fine chemical
industries.3,4
Lipases (E.C. 3.1.1.3) and esterases (E.C. 3.1.1.1) are the
main groups of natural biocatalysts that promote ester bond
cleavage and formation.5 The biological role of lipases is
probably digestive and of esterases is not completely known.6
All aspects described above demonstrate how
fundamentally significant are lipases and esterases for
biotechnological processes. However, the search for new
biocatalysts requires rapid methods to detect specific and
enantioselective enzymes.
*e-mail: anita@iqm.unicamp.br

Vol. 21, No. 8, 2010
Mantovani et al.
1485
Several methodologies have been applied to screen
hydrolases11 however the fluorogenic substrates coupled
to umbelliferone are stable at high temperature and over
a broad range of pH-values12 and have been reported as
screening probes for epoxide hydrolases13 and Baeyer-
Villiger monooxygenases,14 using microorganism whole
cells. This methodology has been applied to the screening
of a great number of activities in strain collections.13,14 The
general principle of the method involves a sequence of steps
in which a fluorescent probe is released after enzymatic
transformation (Scheme 1).
were monitored for esterases using fluorogenic probes and
most active bacterial strains were identified by sequencing
their 16S rRNA genes.
For screening assays, the bacterial strains were grown
in appropriate conditions in slant tubes using nutrient broth
(NB)15 culture medium for 48 h at 30 °C. The colonies
developed on the agar surface were removed and transferred
to previously weighed eppendorf tubes (wet colonies). The
cells were resuspended in 100 mmol L-1 pH 8.8 borate
buffers in order to prepare 0.2 mg mL-1 suspensions.
For scale up bioreaction assays the cells were grown in
500 mL erlenmeyer flasks containing 200 mL of the NB
culture medium with continuous shaking (150 rpm) during
3-4 days at 30 °C. After this stage, the cells were harvested
by centrifugation (5000 rpm, 10 min and 18 °C) and used
in the biocatalytic reactions.
Identification of bacterial strains
The identifications were performed at CPQBA/
Unicamp under the supervision of Dr. Valeria Maia de
Oliveira. After bacterial growth on agar plates, genomic
DNA of pure cultures was isolated according to the
protocol described by Pitcher et al.16 PCR amplification
and sequencing of partial 16S rDNA fragments were
carried out as described by Rodrigues et al.17 Identification
was achieved by comparing the sequences obtained with
16S rRNA sequence data from type strains available at
the public databases Genbank (http://www.ncbi.nem.nih.
gov) and RDP (Ribosomal Database Project, Wiscosin,
USA, http://www.cme.msu.edu/RDP/html/index.html).
The sequences were aligned using the CLUSTAL X
program18 and analyzed using MEGA software v. 2.1.19
Evolutionary distances were derived from sequence-pair
dissimilarities, calculated as implemented in MEGA using
the DNA substitution model reported by Kimura,20 and the
phylogenetic tree was prepared using the neighbor-joining
(NJ) algorithm21 with bootstrap values calculated from 1000
replicate runs.
Scheme 1. Fluorogenic assay representation to detect esterases and
lipases.12
Herein we describe the application of fluorogenic
probes coupled to umbelliferone to screen hydrolases in
soil microorganisms using whole cells. The best hits were
validated with non-fluorogenic substrates.
Experimental
General methods
Merck 60 silica gel (230-400 mesh ASTM) was used
for flash chromatography. Dichloromethane was dried
over CaH2 and distilled immediately before use. 1H NMR
(300.01 MHz, CDCl3) and 13C NMR (75.50 MHz, CDCl3)
spectra were recorded on a Gemini 300P from Varian
Instruments using (CH3)4Si as internal reference (d 0.00).
Chemical shifts d are given in ppm and coupling constants
J are given in hertz. GC-MS analyses were performed with
an Agilent 6890 Series GC System and mass spectra were
recorded with a Hewlett-Packard 5973 Mass Selective
Detector (70 eV) using a HP-5MS - crosslinked 5%
phenyl methyl siloxane (30 m × 0.25 mm i.d. × 0.25 µm
film thickness) fused silica capillary column with helium
as carrier gas (1 mL min-1). Chiral GC was performed
on Agilent 6850 Series GC System with a FID detector,
using hydrogen as a carrier gas (10 psi) and a Chirasil-Dex
CB b-cyclodextrin chiral column (Chrompack, 25 m ×
0.25 mm i.d. × 0.25 µm film thickness).
Fluorescence measurements
Enzymatic activities were investigated with fluorogenic
substrates, 4-[(2-oxo-2H-1-benzopyran-7-yl)oxy]butane-
1,2-diyldiacetate (1), 4-[(2-oxo-2H-1-benzopyran-7-yl)
oxy]butane-1,2-diyl dipropionate (2) and 4-[(2-oxo-2H-1-
benzopyran-7-yl)oxy]butane-1,2-diyldioctanoate(3). These
fluorogenic probes were appropriate to search for esterases
in the miniaturized format using 200 µL 96-well microtiter
plates and were synthesized according to the procedure
of Reymond and co-workers.22 Hydrolytic activities were
screened using substrates 1, 2, 3 (10 µL, 2.0 mmol L-1)
(Scheme 1), microbial cells (100 µL, 0.5 mg mL-1) in borate
Microorganisms
Bacteria isolated from Silves soils (Amazonas State,
Brazil, 1 to 36) and from Ilhéus (Bahia State, Brazil, 37-64)

1486
Esterase Screening using Whole Cells of Brazilian Soil Microorganisms
J. Braz. Chem. Soc.
buffer (20 mmol L-1, pH 8.0); BSA (80 µL, 5.0 mg mL-1)
and NaIO4 (10 µL, 20 mmol L-1). The positive control for
hydrolases was prepared using diol 4 (10 µL, 2.0 mmol L-1),
BSA (80 µL, 5.0 mg mL-1), microbial cells (100 mL,
0.5 mg L-1) and NaIO4 (10 µL, 20 mmol L-1) and the
negative control using the substrates (10 µL, 2.0 mmol L-1),
BSA (80 µL, 5.0 mg mL-1), pH 8.0 borate buffer (100 µL,
20 mmol L-1) and NaIO4 (10 µL, 20 mmol L-1). These assays
were monitored for 10 h at 30 °C.
All the assays and controls were measured in duplicate
andanalyzedinafluorescencespectrophotometer(Flashscan
530Analytic Jena - lex 460 nm). The fluorescence intensity
increase in the system was related to the umbelliferone
released into the reaction media. All the results obtained
correlate the average values between duplicate readings
minus the average value for the negative control, which
express the substrate tendency for spontaneous hydrolysis.
The conversion values were estimated based on the
maximum emission of the umbelliferone anion fluorescence
at pH 8.0 for each assay in comparison with the positive
control for each microorganism. Only those assays that
resulted in 5% conversion value (about 100 fluorescence
units) were considered positive.
dipropanoyl esters) that is characteristic of esterases
(Table 1).
Table 1. Enzymatic assays to detect hydrolases in soil bacterial strains
with fluorescent probes
Strainsa
Fluorescence intensity / conversion (%)
probe 1
probe 2
probe 3
18
19
21
23
27
28
31
32
33
34
44
47
52
53
59
61
146 / 10
1923 / 128
114 / 8
135 / 9
1618 / 108
318 / 21
216 / 14
530 / 35
-
-
-
-
-
104 / 7
149 / 10
-
-
-
-
-
-
1421 / 95
1957 / 130
117 / 8
107 / 7
1331 / 89
-
-
-
-
318 / 21
111 / 7
110 / 7
92 / 6
-
-
-
311 / 21
-
1128 / 75
118 / 8
-
-
-
100 / 7
96 / 6
-
65 / 4
Biocatalytic reactions for hydrolases
a 1-36 Refer to microorganisms isolated from Silves soil (Amazonas State,
Brazil) and 37-68 refer to microorganisms isolated from Silves soil from
Ilhéus (Bahia State, Brazil).
A 125 mL erlenmeyer flask containing 40 mL of
pH 7.0 phosphate buffer and wet whole cells (2-4 g)
were stirred at 150 rpm and maintained at 28 °C. Then,
20 mL of the substrates 1-phenylethanol acetate (5),
1-phenylethanol propionate (6) and 1-phenylethanol
octanoate (7) (synthesized as described in the electronic
Supplementary Information) were added to the medium
(final substrate concentration 0.5 mL mL-1). Bioconversion
was monitored from time to time by extracting aliquots
from the bioreaction with ethyl acetate after saturation
with NaCl. Conversion was based on residual substrates
present in a 0.5 mL sample extracted with ethyl acetate
(0.5 mL) containing an internal standard (benzophenone,
0.01 mg mL-1) and determined by GC-MS.
Considering these assays we can classify these
microorganisms into four groups: those best for acetate
hydrolyses (18, 19, 27, 28, 47 and 53) and those strains
best for propionate hydrolyses (33, 59 and 61). A third
group (21, 23, 31, 32, 34, and 44) hydrolyzes both acetate
and propionate.
The last group, with strains 52 and 61, is active on
longer chain acyl esters such as octanoyl. The strain 52
revealed increasing hydrolytic activity with increasing acyl
chain length as do lipases (Figure 1).
These results mainly indicate the presence of esterase
producers among the evaluated microorganisms and it can
be observed that, as a rule, they promote the hydrolysis of
short chain esters such as acetate and propionate.6,23 For
octanoyl ester 3 only few of the microorganisms promoted
meaningful hydrolysis of this probe, probably because it is
more adequate to test lipases that require a higher level of
lipophilicity in the substrate for a good reactivity.
Results and Discussion
Whole cell screening using fluorogenic probes
High throughput screening assays were used to evaluate
hydrolase activity in 64 strains, revealing that 16 strains
showed biocatalytic activity, confirming the ubiquity
associated to lipases and esterases as most microorganisms
promoted the hydrolysis of probes 1, 2 or 3.
Non-fluorogenic compound assays
Inaddition,theseresultsrevealedthat13microorganisms
tested showed hydrolytic preference for short-length acyl
chains, as presented in substrates 1 and 2 (diacetyl and
The fast screening methodology described above
allowed the detection of several microorganisms possessing
chemoselective esterases active on acetate, propanoate

Vol. 21, No. 8, 2010
Mantovani et al.
1487
These microorganisms were also active against oleates
but only at low conversion rate, which is, in fact, in perfect
agreement with the results observed with the fluorogenic
probes, where with monitoring probe 3 we could observe
low values of fluorescence intensity (Table 2). The
same is true with Bacillus strains in general which are
usually as regarded as good hydrolases producers with
higher hydrolytic activity on short and medium size acyl
chain lengths and are considered by the Food and Drug
Administration of USA (FDA) as safe organisms.23-26
These experiments confirm the substrate selectivity
observed in screening assays and the significantly different
enzymatic behavior depending on the fatty acid chain
length of the esters chosen as probes. Consequently this
methodology can be applied to rapidly evaluate large
microorganisms libraries with reliable results.
Figure 1. Chemoselectivity group classifications based on esterase
activities on various fluorogenic probes 1-3 of diverse chain length.
and octanoate derivatives. These results were validated
using non-fluorogenic compounds to access the enzymatic
conversion range and as well as the enantioselectivity. For
this purpose we selected 1-phenylethanol acyl derivatives
(5, 6 and 7) as model substrates to better evaluate strains 19,
32, 44, 53 and 59, previously selected in the miniaturized
assays with fluorescent probes as possessing good
enzymatic activity.
Table 2. Substrate selectivity with fluorescent and non-flluorescent probes
All selected microorganisms were identified by 16S
rRNA and belong to the Bacillus genus. Their hydrolytic
enzymes converted esters 5, 6 and 7 to the alcohol 8
(Scheme 2, Table 2) confirming the previous enzymatic
activity detected with the fluorescent probes.
In addition, Bacillus sp. Cohn 1872 (strain 19)
preferentially hydrolyzed acetyl > propanoyl > octanoyl
ester derivatives as observed in the HTS experiments,
keeping the same priority towards the non-fluorogenic
1-phenylethanol acyl esters. Bacillus sp. Cohn 1872
(strain 32) and Bacillus sp. Cohn 1872 (strain 44) also
displayed similar HTS behavior promoting preferential
hydrolysis of the propanoate derivative.
Conversion %
Dt c = 50 % (h)b
(fluorescent signal)a
Microorganisms
1
Bacillus sp. Cohn 1872 (19) 77
Bacillus sp. Cohn 1872 (32) 21
Bacillus sp. Cohn 1872 (44) 4.5
2
3
0
-
5
6
7
2.5
78
53
4.0
2.0
14
> 24
1.5 > 24
-
2.0 0.75 > 10
a Conversion value obtained from HTS experiments; b time needed for
50% conversion of non-fluorogenic substrates using 2.0 g of wet cells in
50 mL of 20 mmol L-1 phosphate buffer, pH 7.0 with 20 mg of substrate.
Using 5, 6 and 7 as substrates we could also estimate the
enantioselectivityratio(E)ofthemicroorganismsselectedin
HTS. The enantiopreferences of these microorganisms were
determined by GC-FID using a chiral column and focusing
on the enantiomeric excess of the produced phenylethanol
8 (Table 3). The optimum analytical condition for the ( )-8
resolution and identification, where R-8 had the shorter
retention time, was confirmed by coinjection of ( )-8
with enantiomerically pure R-8 (Figure 2). These results
are summarized in Table 3. Microorganisms Bacillus sp.
O
O
esterase or
lipase
O
R
OH
O
R
+
5 R=CH3
8
6 R=C2H5
7 R=C7H15
Scheme 2. Kinetic resolution of phenylethanol derivatives 5, 6 and 7.
Table 3. Ester hydrolysis of 5 and 6 using the selected microorganisms
Absolute
configuration
Microorganisms
Substrate
Conversiona %
eeb %
Ec
Bacillus sp. Cohn 1872 (19)
6
6
48
50
54
87
89
99
96
51
60
R
R
R
Bacillus sp. Cohn 1872 (44)
Bacillus sp. Cohn 1872
(Bacillus cereus) (53)
5
5
32
55
43
99
34
49
R
R
Bacillus subtilis subsp. subtilis
(Ehrenberg 1835) Cohn 1872 (59)
aDetermined by the average ratio between product area and the area for internal standard; bEnantiomeric excess, ee = ((A-B) / (A+B))×100; cEnantiomeric
ratio estimated for substrate, E = ln[(1-c) (1-ee)] / ln[(1-c) (1+ee)].

1488
Esterase Screening using Whole Cells of Brazilian Soil Microorganisms
J. Braz. Chem. Soc.
Cohn 1872 (44), Bacillus sp. Cohn 1872 (Bacillus cereus)
(53) and Bacillus subtilis subsp. subtilis (59) were able to
convert the acetyl derivative 5 into phenylethanol 8 with
significantly high E values (E > 30).
Regarding the hydrolysis of the propionate derivative
6, the highest enantioselective ratios were observed for
Bacillus sp. Cohn 1872 (19) (E = 96), and Bacillus sp.
Cohn 1872 (44) (E = 51). It is interesting to note that all
microorganisms showed preference for the R-enantiomer
in agreement to the Prelog rule extension.27,17
has to be aware that the miniaturized and the HTS assays
can provide false positives as well as false negatives but
has the advantage of testing a considerable number of
microorganisms in short periods of time. Microorganisms
showing high E values of 1-phenylethanol derivative
hydrolysis can be applied to produce enantiomerically
pure esters which represents a key argument in favor of
their conservation. Consequently the strains were freeze
dried and also kept in glycerol in our private collection.
Strains 44, 53 and 59 will also be deposites at the
CBMAI/CPQBA/Unicamp. Ongoing studies with cell
immobilization and enzyme isolation will be published
in due course.
Supplementary Information
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as pdf file.
Acknowledgments
The authors are grateful for financial support from
Fundação de Amparo a Pesquisa do Estado de São Paulo
(FAPESP), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
We also thank Prof. Carol Collins for helpful suggestions
in style and grammar.
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Web Release Date: April 12, 2010
FAPESP has sponsored the publication of this article.

J. Braz. Chem. Soc., Vol. 21, No. 8, S1, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Esterase Screening Using Whole Cells of Brazilian Soil Microorganisms
Simone M. Mantovani, Luciana G. de Oliveira and Anita J. Marsaioli*
Instituto de Química, Universidade Estadual de Campinas, CP 6154,
13084-971 Campinas-SP, Brazil
Synthesis of the alcohol 8 and hydrolytic substrates 5, 6
and 7
104 (90), 77 (34), 43 (42). 1H NMR (300.01 MHz, CDCl3)
d 1.55 (d, 3H, J 6.6 Hz, H-8); 2.10 (s, 3H, H-10); 5.90 (q,
1H, J 6.6 Hz, H-7); 7.40 (m, 5H, H-2, H-3, H-4, H-5, H-6).
13C NMR (75.5 MHz, CDCl3, dCDCl3 77.0) d 21.3 (CH3,
C-10); 22.2 (CH3, C-8); 72.3 (CH, C-7); 126.0 (2 CH, C-3
and C-5); 127.8 (CH, C-4); 128.5 (2 CH, C-2 and C-5);
141.67 (C0, C-1); 170.3 (C0, C-9).
Alcohol 8
To a round bottom flask containing acetophenone
(1.063 g, 8.71 mmol) in MeOH (3.0 mL), NaBH4 (440 mg,
9.5 mmol) was added and the resulting suspension was
stirred for 20 min. The reaction mixture was treated with 3
portions of 5 mL of brine and the organic phase was dried
over anhydrous Na2SO4 and evaporated. Purification of the
crude product by flash column chromatography (hexanes/
EtOAc, 3:4) afforded 8 as a colorless oil, in 87% yield.
MM: 122 g mol-1 (C8H10O). EI-MS m/z 122 (M.+, 34%), 107
(100), 79 (90), 77 (51), 51 (14), 43 (18). 1H NMR (300.01
MHz, CDCl3) d 1.45 (d, 3H, J 6.6 Hz, H-8); 2.10 (sl, 1H,
OH); 4.90 (q, 1H, J 6.6 Hz, H-7); 7.40 (m, 5H, H-2, H-3,
H-4, H-5, H-6). 13C NMR (75.5 MHz, CDCl3, dCDCl3 77.0)
d 25.0 (CH3, C-8); 69.8 (CH, C-7); 125.3 (2 CH, C-3 and
C-5); 127.0 (CH, C-4); 128.0 (2 CH, C-2 and C-6).
Ester 6: Purification of the crude product by flash
column chromatography (hexanes/EtOAc, 5:1) afforded 6
as a colorless oil, in 80% yield. MM: 178 g mol-1 (C11H14O2).
EI-MS m/z 178 (M.+, 25%), 122 (95), 105 (100), 104 (78),
1
77 (26), 57 (31). H NMR (300.01 MHz, CDCl3) d 1.17
(t, 3H, J 7.7 Hz, H-11); 1.50 (d, 3H, J 6.7 Hz, H-8); 2.35
(q, 2H, J 7.7 Hz, H-10); 5.90 (q, 1H, J 6.7 Hz, H-7); 7.40
(m, 5H, H-2, H-3, H-4, H-5, H-6). 13C NMR (75.5 MHz,
CDCl3, dCDCl3 77.0) d 9.06 (CH3, C-11); 22.2 (CH3, C-8);
27.8 (CH2, C-10); 72.0 (CH, C-7); 126.0 (2 CH, C-3 and
C-5); 127.7 (CH, C-4); 128.4 (2 CH, C-2 and C-5); 141.8
(C0, C-1); 173.6 (C0, C-9).
Syntheses of esters 5, 6 and 7, general procedure:
To a solution of alcohol 8 (1.617 g; 13.2 mmol)
in methylene chloride (25 mL) at 0 °C, acyl chloride
(21.2 mmol of acetyl chloride or propanoyl chloride
or octanoyl chloride) and DMAP (1.58 g, 16.8 mmol)
were added. The reaction was stirred for 10 h at room
temperature. After washing with a saturated solution of
NaHCO3, the organic phase was dried over Na2SO4 and
evaporated under reduced pressure to afford the expected
products 5, 6 or 7.
Ester 7: Purification of the crude product by flash
column chromatography (hexanes/EtOAc, 5:1) afforded 7
as colorless oil, in 79% yield. MM: 248 g mol-1 (C16H24O2)
EI-MS m/z 248 (M.+, 2%), 143 (5), 122 (100), 105 (87), 57
(11). 1H NMR (300.01 MHz, CDCl3) d 0.86 (t, 3H, J 6.0
Hz, H-16); 1.20-1.35 (m, 8H, H-12, H-13, H-14 and H-15),
1.52 (d, J 6.0 Hz, 3H, H-8); 1.62 (t, 2H, J 6.0 Hz, H-11);
2.32 (t, 2H, J 6.0 Hz, H-10); 5.90 (q, 1H, J 6.0 Hz, H-7);
7.22-7.40 (m, 5H, H-2, H-3, H-4, H-5, H-6). 13C NMR
(75.5 MHz, CDCl3, dCDCl3 77.0) d 13.9 (CH3, C-16), 22.2
(CH3, C-8); 22.5 (CH2, C-15), 24.9 (CH2, C-14), 28.8 (CH2,
C-13); 28.9 (CH2, C-12); 31.6 (CH2, C-11); 35.5 (CH2,
C-10); 71.9 (CH, C-7); 125.9 (2 CH, C-3 and C-5); 127.7
(CH, C-4); 128.4 (2 CH, C-2 and C-5); 141.8 (C0, C-1);
173.0 (C0, C-9).
Ester 5: Purification of the crude product by flash
column chromatography (hexanes/EtOAc, 9:1) afforded 5
as a colorless oil, in 85% yield. MM: 164 g mol-1 (C10H12O2).
EI-MS m/z 164 (M.+, 22%), 122 (100), 107 (37), 105 (74),
*e-mail: anita@iqm.unicamp.br