Biocatalysis: Fungi mediated novel and selective 12β- or 17β-hydroxylation on the basic limonoid skeleton

Article abstract of DOI:10.1039/c3gc40193f

Basic limonoids carrying a 4,4,8-trimethyl-17-furanylsteroid skeleton are a class of triterpenoids and well-known for their insecticidal as well as a vast array of pharmacological activities. Rare and synthetically challenging 12β- and 17β-hydroxylation was achieved on the basic limonoid skeleton to produce a novel series of hydroxylated limonoids using fungi-mediated biocatalysis. The fungal system belonging to the genera of Mucor efficiently converted azadiradione, epoxyazadiradione, gedunin and their derivatives into corresponding 12β- and/or 17β-hydroxy derivatives. The position and stereochemistry of hydroxylation was determined by rigorous spectroscopic and crystallographic studies. This fungi-mediated stereo- and regio-selective hydroxylation process was highly efficient and mild enough to sustain chemically sensitive functional groups around the basic limonoid skeleton. Modifications of specific functional groups and variation in biocatalyst were shown to bring selectivity among 12β- or 17β-hydroxylation.

Full text of DOI:10.1039/c3gc40193f

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Biocatalysis: fungi mediated novel and selective 12β-
or 17β-hydroxylation on the basic limonoid skeleton†‡
Cite this: Green Chem., 2013, 15, 1311
Saikat Haldar,a Swati P. Koleta and Hirekodathakallu V. Thulasiram*a,b
Basic limonoids carrying a 4,4,8-trimethyl-17-furanylsteroid skeleton are a class of triterpenoids and well-
known for their insecticidal as well as a vast array of pharmacological activities. Rare and synthetically
challenging 12β- and 17β-hydroxylation was achieved on the basic limonoid skeleton to produce a novel
series of hydroxylated limonoids using fungi-mediated biocatalysis. The fungal system belonging to the
genera of Mucor efficiently converted azadiradione, epoxyazadiradione, gedunin and their derivatives
into corresponding 12β- and/or 17β-hydroxy derivatives. The position and stereochemistry of hydroxy-
lation was determined by rigorous spectroscopic and crystallographic studies. This fungi-mediated stereo-
and regio-selective hydroxylation process was highly efficient and mild enough to sustain chemically sen-
sitive functional groups around the basic limonoid skeleton. Modifications of specific functional groups
and variation in biocatalyst were shown to bring selectivity among 12β- or 17β-hydroxylation.
Received 24th January 2013,
Accepted 5th March 2013
DOI: 10.1039/c3gc40193f
www.rsc.org/greenchem
their derivatives come under this group (Fig. 1). The strong
antifeedant properties1 along with anti-plasmoidal,10 anti-
Introduction
Limonoids, chemically classified as tetranortriterpenoids, are HIV,11 and anti-inflammatory12 activities of azadiradione and
metabolically modified triterpenes having an intact 4,4,8-tri- epoxyazadiradione have attracted the attention of synthetic
methyl-17-furanylsteroid precursor skeleton (basic limonoid) chemists in the last two decades.13,14 In particular, gedunin
or further rearranged and highly oxygenated construction, is a well-studied anti-malarial,15 anti-carcinogenic16 and anti-
creating a structural diversity.1 Their existence is mainly con- ulcerogenic agent.17
fined to the plants of the families Meliaceae (e.g. Neem etc.)
The potency of limonoids as biologically active entities
and Rutaceae (e.g. Citrus etc.).2 Although the first isolated can be varied with the modification of functional groups
limonoid ‘limonin’ (from which the term ‘limonoid’ is ornamentally arranged around their skeletons. According to
derived) disclosed the bitter characteristic of navel orange
juice in 1949,3 limonoids only came into the real picture when
they were found to possess strong antifeedant and growth-inhi-
biting properties towards pests, mainly neem limonoids, and
in specific Azadirachtin A.4,5 Subsequently, they have been
found to possess anti-cancer,2,6 anti-malarial,7 anti-HIV,8 anti-
microbial9 and several other pharmacological activities. Apart
from C-seco limonoids, the basic precursors carrying an ‘intact
apo-euphol skeleton’ with 14,15-β-epoxide and/or a cyclohexe-
none (3-oxo-1-ene) A ring also showed similar bioactivities.1
Azadiradione (1), epoxyazadiradione (10), gedunin (19) and
aCSIR-National Chemical Laboratory, Department of Organic Chemistry,
Pune-411008, India. E-mail: hv.thulasiram@ncl.res.in; Fax: +91 2025902636;
Tel: +91 2025902478
bCSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi-110007,
India
†Electronic supplementary information (ESI) available: Synthetic procedures,
copies of NMR, MS spectra and their analysis. CCDC 918564. For ESI and crystal-
lographic data in CIF or other electronic format see DOI: 10.1039/c3gc40193f.
‡This work is dedicated to Prof. K. N. Ganesh, IISER, Pune, on the occasion of
Fig. 1 Basic limonoid skeleton and representative members of the basic limo-
his 60th birthday.
noid family.
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Champagne et al., the limonoid biosynthetic pathway is
featured by evolution towards increasing rearrangement and
oxidation of the basic skeleton to produce highly oxygenated
terpenoids which are biosynthesized for self-defense against
insects.1 Thus, increasing oxidation and rearrangement can
lead to enhanced activity. But conventional synthetic tools are
sometimes tedious and time consuming for desired functional
group modifications on the ‘active structural unit’ due to the
presence of inactive carbons, steric hindrance, sensitivity of
functional groups towards external reagents and the presence
of identical groups with similar reactivity. Frequently, syn-
thesis involves several protection–deprotection steps which
make the overall process low-yielding. On the other hand, bio-
catalysis is a well-established ‘green’ technique for carrying out
stereo- and regio-selective functionalization of various sen-
sitive and complex molecules in mild reaction conditions.18–21
The insecticidal properties along with the diverse pharmaco-
Fig. 2 Time-course experiment in graphical representation (substrate concen-
tration: 0.2 g l−1 medium): ( ) azadiradione; ( ) 17β-hydroxyazadiradione; (
12β-hydroxyazadiradione.
●
■
▲
)
logical activities of basic limonoids directed us to screen control) (ESI†). Resting cell experiment further confirmed that
various fungal systems for novel and useful biotransformations the hydroxylase system involved in the transformation is con-
like C–H oxidation on synthetically inactive carbons.
stitutive. Time-course experiment with the same substrate con-
In this article we have depicted systematically the scope of centration clearly showed that M881 quantitatively converted 1
whole cell biocatalysis for the production of 17β- and into two metabolites after 8 days of incubation period (Fig. 2).
12β-hydroxy metabolites of basic limonoids with excellent con-
version. Indeed, it is the first report on the biotransformation
of an intact limonoid skeleton furnishing novel stereo- and
regio-selective hydroxylated metabolites which would be chal-
lenging to achieve by chemical synthetic methods. The ability
of a specific biocatalyst to transform a variety of structurally
modified basic limonoids in a highly efficient manner was
explored, which broadened the substrate scope within the
basic limonoid family. More interestingly, either of the 17β- or
12β-hydroxylation was achieved selectively by varying the bio-
catalyst or the skeletal functional groups.
Preparative scale fermentation and purification
The experiment was further taken ahead in a preparative scale
with an aim of purification and characterization of the meta-
bolites. Substrate concentration studies (0.1 to 0.4 g l−1) indi-
cated that the fungal system M881 quantitatively transformed
1 into 2 and 3 at the concentration of 0.2 g l−1 with 8 days of
incubation. An increase in the substrate concentration (>0.2 g
l−1) considerably decreased the rate of bioconversion. The opti-
mized substrate concentration (0.2 g l−1) and incubation
period (8 days) as evidenced from time-course experiment were
used for the preparative scale fermentation with 0.4 g of aza-
diradione. The purification, using a silica gel column, of the
extracted crude biotransformation products (experimental
section) obtained by incubating azadiradione (1) with M881
furnished two pure metabolites characterized as 17β-hydroxy-
azadiradione (2, 0.21 g, yield ∼51%, Rf: 0.65, Rt: 38.0 min) and
12β-hydroxyazadiradione (3, 0.14 g, yield ∼33%, Rf: 0.56, Rt:
28.1 min) (Scheme 1).
Results and discussion
Screening and time-course experiment
Azadiradione (1), an isolated limonoid from the fruit of
Azadirachta indica (Neem), was used as the representative
model substrate for the screening experiment at an analytical
scale. Over twenty five different species of filamentous fungi
belonging to the genera Aspergillus, Mucor, Cephaliophora,
Fusarium, Rhizopus and Gibberella were screened for their
Characterization of metabolites
ability to carry out useful transformations of azadiradione. Metabolites were structurally characterized on the basis of IR,
1
Screening experiments were carried out with 0.2 g l−1 of sub- LC-ESI-MS (positive ion mode), HRMS, H, 13C, DEPT and 2D
strate concentration with an 8 day incubation period at 30 °C (COSY, NOESY, HMBC, HSQC) NMR studies. The HRMS ana-
and 180 rpm. TLC, HPLC and LCMS analyses of the extracted lysis of metabolite 2 showed an ion peak at m/z 489.2254
biotransformed products indicated that one of the fungal (C28H34O6Na), indicating the addition of an oxygen atom to
systems belonging to the genera Mucor (National Collection of the azadiradione structure (C28H34O5). Appearance of a broad
Industrial Microorganisms or NCIM, Pune, catalogue no. 881 absorption band at 3448 cm−1 in the IR spectrum and a highly
and abbreviated as M881) could quantitatively carry out the abundant peak at m/z 449.2 ([M − OH]+) in the LC-ESI-MS of 2
transformation and hence was selected for fermentation at the indicated the presence of a hydroxyl group. From the 13C
preparative scale. Bioconversion was validated by comparing (DEPT) NMR studies, the number of quaternary carbons
with two control experiments: organism without substrate increased by one with a loss of one tertiary carbon in compari-
(organism control) and substrate without organism (substrate son with the substrate, which suggested that hydroxylation
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Fig. 4 ORTEP diagram of 12β-hydroxygedunin (20).
from 15.83 to 28.31 and 30.36 to 68.37 ppm, respectively,
accompanied by a downfield shift of quaternary C-13 carbon.
These observations indicated the presence of the hydroxyl
group at the C-12 position, which was again confirmed by
HMBC correlation of hydroxylated carbon (C-12) with H-17, 18
and H-12 with C-18, 9, 13, 14. Hydroxylation was found to be
equatorial (on the β-face) on the basis of strong NOESY cross
peaks H-12/H-18, H-11α, H-22 (Fig. 3). The presence of axial
hydrogen on C-12 was also supported by the high coupling
constant (7.32 Hz) of the broad doublet assigned for H-12. The
position and stereochemistry of 12β-hydroxylation was further
supported by the analysis of single crystal X-ray diffraction
data obtained for the metabolite of gedunin (Fig. 4).
Scheme 1 Biotransformation pathway for azadiradione (1) by Mucor sp.
M881.
occurred on a tertiary carbon. The 1H NMR spectra of 2 was
similar to that of 1 except the absence of the peak at 3.43 ppm
for 17-H. 13C NMR showed a downfield shift of C-17 from
60.76 to 80.56 ppm suggesting the presence of a hydroxyl
group at C-17. HMBC analysis showed the correlation of
hydroxylated carbon (C-17) with H-15, 18, and 22 which
further confirmed the 17-hydroxylation. The NOESY spectrum
containing the cross peaks H-21/H-18 and H-22/H12α, evi-
dence that the furan ring is in the α-orientation (Fig. 3). Based
on these spectral characteristics, the metabolite was identified
as 17β-hydroxyazadiradione (2). Also, the 1H and 13C NMR data
matched well with the earlier report.22
Similarly, from the HRMS data of metabolite 3, the mole-
cular formula was established as C28H34O6 based on the ion peak
at m/z 489.2286 (C28H34O6Na) and the presence of a hydroxyl
group was confirmed by the appearance of a broad absorption
band at 3448 cm−1 in the IR spectrum and a highly abundant
peak at m/z 449.2 ([M − OH]+) in LC-ESI-MS. Loss of a methyl-
ene carbon and an increase in the number of tertiary carbons
in the 13C (DEPT) NMR of the metabolite, indicated that the
hydroxylation occurred on a methylene carbon. Among the
three methylene carbons (C-6, 11 and 12) in the structure,
the chemical shift of C-6 remained almost unchanged in the
metabolite whereas C-11 and C-12 underwent a downfield shift
Effects of functional groups on bioconversion profile
To further investigate the substrate specificity of the organism
within the basic limonoid family, the biotransformation was
studied with seven natural or semi-synthetic derivatives of aza-
diradione (Table 1). Fermentation was carried out at the pre-
parative scale under similar conditions for each individual
azadiradione derivative and the metabolites were characterized
(ESI†) after purification.
Reduction and epoxidation of the double bond in the A
ring of azadiradione did not affect the ability of the micro-
organism to carry out hydroxylation at the 17β- and 12β-
positions as observed with 1,2-dihydroazadiradione (4)
and 1,2α-epoxyazadiradione (7). Both the substrates were con-
verted to the 17β- and 12β-hydroxy metabolites almost in the
same ratio (3 : 2), like azadiradione, with excellent conversion
rates. These results indicated that the enone moiety in the ring
A is not critical for carrying out the transformation. It is
interesting to note that the organism specifically carried out
hydroxylation at the 12β-position on epoxyazadiradione (10)
and its double bond reduced derivative, 1,2-dihydroepoxyaza-
diradione (12). Indeed, the organism completely failed to
carryout the 17β-hydroxylation on 10 and 12 as indicated by HPLC
and LC-MS analyses. The organism also accepted gedunin (19,
expanded D ring as six membered lactone) as a substrate
to produce 12β-hydroxy gedunin as a sole biotransformed
product. The absolute structure of 12β-hydroxygedunin was
Fig. 3 Key HMBC (H
and 3.
C) and NOESY (H
H) correlations of compounds 2
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Table 1 Selectivity in biotransformation products with the variation in functional groups on the limonoid skeleton
Metabolites
Entry
no.
Time
(days)
Conc.
Substrate
Biocatalyst
(g l−1
)
% Conversion
99
17β-Hydroxy metabolite
%
12β-Hydroxy metabolite
%
1
2
M881
8
0.2
61
38
M881
M881
M881
M881
M881
M881
M881
8
0.2
0.2
0.2
0.2
0.2
0.2
0.2
99
96
99
93
92
94
96
59
57
—
—
71
—
—
40
39
99
93
—
94
96
3
4
5
6
7
8
8
8
8
8
8
8
Not produced
Not produced
Not produced
Not produced
Not produced
a Metabolites are novel i.e. synthesized or isolated for the first time.
1314 | Green Chem., 2013, 15, 1311–1317
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elucidated by single crystal X-ray diffraction analysis23 along overall 75.4% conversion (Table 2). Therefore, these systems
with NMR studies. Similarly, the organism was able to hydroxy- can be used for the selective 17β-hydroxylation.
late 7-deacetylepoxyazadiradione (17) at the 12β-position as a
single metabolite. However, when 7-deacetylazadiradione (14,
nimbocinol) was used as a substrate, the position of hydroxy-
lation was changed to the 17β-position to produce 17β-hydroxy-
Conclusions
nimbocinol (15, 71%) along with the oxidised product 7-oxo- In conclusion, we have developed a highly efficient fungi-
17β-hydroxynimbocinol (16, 21%, ESI†) as metabolites. These mediated bioconversion technique for the 12β- and 17β-
results suggested that two functionalities (epoxy group in ring hydroxylation of the basic limonoid family. The synthetically
D and acetate in ring B) were critical for carrying out the challenging hydroxylation on inactive carbon (C-12) was
hydroxylation and may bring about a sharp alternation in the achieved in a highly efficient and stereoselective manner.
product distribution ratio (17β-hydroxy : 12β-hydroxy). It is Chemically sensitive functional groups such as lactone,
known that chemically synthesized 17β-hydroxynimbocinol epoxide or ester were tolerable to the mild reaction condition.
(15) (∼10% yield) from azadiradione (1) showed two-fold Using the biocatalyst M881, a variety of 12β- and 17β-hydroxyl-
potency against Anopheles stephensi Liston compared to imonoids were generated among which many are hitherto
nimbocinol.24 Herein, the biocatalyst M881 was able to oxidize unknown (3, 5, 6, 8, 9, 11, 13, 18 and 20) and can prove to be
nimbocinol (14) to a more potent insecticide 17β-hydroxy- potent bioactive molecules. Actually, 12β-hydroxy natural pro-
nimbocinol (15) under mild and more ecofriendly conditions ducts are rare in nature and therefore this report will create an
with much higher yields.
opening to produce 12β-hydroxy limonoids and evaluate their
Modifications in the skeletal functional groups didn’t bioactivities. The fungal system mimicked the plant monooxy-
change the outcome (such as position and stereochemistry of genase system for the production of 17β-hydroxyazadiradione
hydroxylation) of bioconversion but did influence the selecti- which is one of the minor metabolites present in neem fruit.22
vity between two regioisomers. As evidenced, 14,15β-epoxid- Either of the regio-isomers (12β- and 17β-hydroxyl limonoids)
ation on the basic limonoid skeleton can eliminate was obtained selectively by specific modification of the skeletal
17β-hydroxylation to produce the 12β-hydroxy derivative as the functional groups or varying fungal strains within the genera
single metabolite. Importantly, biocatalyst M881 can be used of Mucor and Rhizopus.
as the general tool for the production of 12β- or 17β-hydroxy
limonoids of the intact limonoid family.
Experimental
General information
Biotransformation of azadiradione (1) by various fungi
Chemicals (GR grade) for preparing media and performing the
reactions were purchased from Merck and Sigma. All the HPLC
chromatograms were recorded on a Waters HPLC system fitted
with a UV-Vis detector and analytical XBridge C18 column
From the screening experiments, we found that the fungal
systems belonging to genera Mucor and Rhizopus have been
able to efficiently and selectively hydroxylate at 12β- and
17β-positions of azadiradione. Analyses of HPLC profile of
screening experiments revealed that some of the Mucor and
Rhizopus species converted azadiradione to 17β-hydroxy as the
major metabolite in fairly good yields (Table 2). For example,
one of the Rhizopus sp. (NCIM catalogue no. 1009) converted
(4.6 × 100 mm). Screening samples were injected in HPLC at a
flow rate of 1 mL min−1 with a gradient solvent programme of:
0 min, 40% methanol–water; 25 min, 50% methanol–water;
40 min, 65% methanol–water; 42 min, 40% methanol–water;
45 min, 40% methanol–water; and monitored at 215 nm.
azadiradione to 17β-hydroxy metabolite in 5 : 1 ratio with an
LC-ESI-MS runs were performed in a Waters Acquity Ultra Per-
formance LC system attached with PDA and Single Quadrupole
Table 2 Product distribution table for the fungal systems showing considerable
detectors. For extractions and preparative chromatography pur-
poses, technical grade solvents were distilled before use and
bioconversion of azadiradione (1)
for HPLC and LC-MS purposes, LC-MS grade solvents were pro-
NCIM catalogue
no.
%
cured from Sigma. Milli-Q water purification system (Millipore,
USA) was used for purifying water required for HPLC and
LC-MS experiments and making fermentation media. Thin-
layer chromatography (TLC) was performed on silica gel
G-coated plates (0.25 mm for analytical) developed three times
in 3% methanol in dichloromethane. Compounds were visual-
ized under UV light (254 nm) or by spraying with a solution of
3.0% anisaldehyde, 2.8% H2SO4, 2% acetic acid in ethanol fol-
lowed by heating for 1–2 min. Purification of compounds was
performed by flash chromatography using 230–400 mesh silica
gel columns and dichloromethane–methanol gradient mixture
Strain
Conversion
% 2 % 3
Rhizopus oryzae
Mucor heimalis
Rhizopus oryzae
Mucor piriformis
Cephaliophora
irregularis
1009
873
75.4
69.3
45.5
45.2
31.6
62.6 12.8
51.6 17.7
877
37.7
37.2
7.8
8.0
a
—
1278
505
17.1 14.5
Aspergillus foetidus
20.4
7.3
4.8
4.7
13.2
7.3
4.8
4.7
7.2
—
—
—
Fusarium proliferatum 1105
Aspergillus niger
Aspergillus giganteus
616
568
a Soil isolated.
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as the eluent. Optical rotations were measured in chloroform into 20 mL of phosphate buffer of pH 7.2 containing 150 mg
at 589 nm on JASCO (P-2000) polarimeter using 10 mm cell dextrose. Then, 3 mg of azadiradione in 0.2 mL acetone was
(c in g 100 mL−1) and thin film IR spectra were recorded with added to the reaction mixture and incubated at 30 °C on a
Perkin Elmer FT-IR spectrophotometer in CHCl3. Recording of rotary shaker (180 rpm) for 36 h. After this incubation period,
NMR (1H, 13C, DEPT-135) data were carried out on a Varian filtered mycelia and broth were extracted with ethyl acetate
INOVA spectrophotometer (200, 400 and 500 MHz) in CDCl3 (20 mL × 2) and analysed by TLC and HPLC.
and the residual solvent or TMS signals were designated as the
(e) Preparative scale fermentation. First, 0.4 g limonoid (in
reference. Mass spectra (MS) were recorded on a MSI autocon- 2 mL acetone) was distributed among 20 flasks, each contain-
cept UK with ionization energy 70 eV and on Waters make ing a well-grown culture of M881 after adjusting the pH of the
QTOF (Synapt-HDMS). Single crystal X-ray data were collected broth to 6.5–7.0. After incubating at 30 °C and 180 rpm for 8
on a Super Nova Dual source X-ray Diffractometer system days, mycelia and broth were separated by filtration using
(Agilent Technologies) equipped with a CCD area detector and muslin cloth and extracted separately with ethyl acetate (2 L ×
operated at 250 W power (50 kV, 0.8 mA) to generate Mo Kα 3 for broth and 300 mL × 3 for mycelia). Both the ethyl acetate
radiation (λ = 0.71073 Å) and Cu Kα radiation (λ = 1.54178 Å). layers were combined and concentrated under reduced
SHELX-9722 was used for the solution of structure.
pressure at 50 °C. Extracted crude product (∼0.5 g) was
purified over silica gel column using a gradient mixture of
dichloromethane and methanol. For example, elution of
Limonoids
Azadiradione and epoxyazadiradione were isolated from neem 230–400 mesh silica gel column (20 × 2.5 cm) loaded with
fruits as reported earlier.12 Derivatives were synthesized follow- 0.53 gm crude biotransformed product of azadiradione
ing the procedure detailed in the ESI.†
furnished 17β-hydroxyazadiradione (2) in 0.6% methanol in
dichloromethane and 12β-hydroxyazadiradione (3) in 0.8%
methanol in dichloromethane.
Microorganisms
The microorganisms used in the present study were obtained
(f) Characterization of the metabolites. The individual
from National Collection of Industrial Microorganisms purified metabolites were characterised by NMR, IR and
(NCIM), NCL, Pune. Mucor piriformis was isolated from garden mass spectra (ESI†) as discussed for the characterization of
soil.25
12β-hydroxy and 17β-hydroxy azadiradione metabolites in the
results and discussion section. 12β-Hydroxygedunin (20) was
crystallized from chloroform (solvent of crystallization = 2) as
General biotransformation procedure
(a) Fermentation conditions. The cultures were stored at colourless needles by slow evaporation of solvent at 4 °C and
4 °C and sub-cultured on PDA (potato-dextrose-agar) slant at its structure was determined by single crystal X-ray diffraction
30 °C. The inoculum prepared from a well-grown (36–48 h) method.
culture was used (3% v/v) for inoculation of 100 mL sterile
modified Czapek Dox medium26 of pH 5.8 in a 500 mL
Erlenmeyer flask. Flasks were incubated at 30 °C on a rotary
shaker (180 rpm) for growing the cultures.
Acknowledgements
(b) Screening experiment. To a well-grown culture (pH of
which was adjusted to 6.5–7.0 using sterile 1 M K2HPO4),
20 mg of azadiradione in 0.2 mL acetone was added and in-
cubated at 30 °C and 180 rpm for 8 days. After this incubation
period, mycelia and broth were extracted separately with ethyl
acetate (three times) and analyzed by TLC and HPLC. All the
screening experiments were analyzed by comparing with
corresponding substrate control and organism control.
(c) Time-course experiment. In time-course experiments,
incubations were carried out for one to ten days using azadira-
dione (0.2 g l−1) in acetone by M881. Aliquots were drawn at
regular intervals of every two days and extracted with ethyl
acetate. The metabolites formed were monitored by HPLC
analysis. The level of different metabolites formed was deter-
mined on the basis of the area under the respective peaks. All
analyses were carried out under identical conditions.
S.H. and S.P.K. acknowledge CSIR, New Delhi for the fellow-
ship. This work is supported by CSIR-New Delhi sponsored
network projects (HCP0002, BSC0124 and CSC0106) and the
Director, CSIR-NCL, Pune.
References
1 D. E. Champagne, O. Koul, M. B. Isman, G. G. E. Scudder
and G. H. N. Towers, Phytochemistry, 1992, 31, 377–394.
2 A. Roy and S. Saraf, Biol. Pharm. Bull., 2006, 29, 191–201.
3 O. H. Emerson, Food Technol., 1949, 3, 248–250.
4 Neem: A Tree for Solving Global Problems, National
Research Council, National Academy, Washington, DC,
1992.
(d) Resting cell experiment. A well-grown culture of M881
(36 h) was filtered and the mycelia obtained were washed
repeatedly with distilled water and phosphate buffer (pH 7.2).
The mycelia were dried by pressing between the filter papers.
Then, 3 mg of mycelia (wet weight) was weighed and added
5 M. Jacobson, Focus on Phytochemical Insecticides: The Neem
Tree, CRC Press, Boca Raton, 1988.
6 T. Tanaka, T. Koyano, T. Kowithayakorn, H. Fujimoto,
E. Okuyama, M. Hayashi, K. Komiyama and M. Ishibashi,
J. Nat. Prod., 2001, 64, 1243–1245.
1316 | Green Chem., 2013, 15, 1311–1317
This journal is © The Royal Society of Chemistry 2013

View Article Online
Green Chemistry
Paper
7 O. Kayser, A. F. Kiderlen and S. L. Croft, Parasitol. Res., 17 V. Lakshmi, N. Singh, S. Shrivastva, S. K. Mishra,
2003, 90, S55–S62.
8 L. Battinelli, F. Mengoni, M. Lichtner, G. Mazzanti,
P. Dharmani, V. Mishra and G. Palit, Phytomedicine, 2010,
17, 569–574.
A. Saija, C. M. Mastroianni and V. Vullo, Planta Med., 2003, 18 A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts
69, 910–913. and B. Witholt, Nature, 2001, 409, 258–268.
9 S. A. M. Abdelgaleil, T. Iwagawa, M. Doe and M. Nakatani, 19 N. Q. Ran, L. S. Zhao, Z. M. Chen and J. H. Tao, Green
Fitoterapia, 2004, 75, 566–572. Chem., 2008, 10, 361–372.
10 G. Chianese, S. R. Yerbanga, L. Lucantoni, A. Habluetzel, 20 T. Ishige, K. Honda and S. Shimizu, Curr. Opin. Chem. Biol.,
N. Basilica, D. Taramelli, E. Fattorusso and O. Taglialatela-
Scafati, J. Nat. Prod., 2010, 73, 1448–1452.
11 J. A. Delafuente, J. J. Marugan, S. S. Cross, A. F. Mateos,
2005, 9, 174–180.
21 C. M. Clouthier and J. N. Pelletier, Chem. Soc. Rev., 2012,
41, 1585–1605.
S. Garcia and A. Menendez, Bioorg. Med. Chem. Lett., 1995, 22 W. Kraus and R. Cramer, Tetrahedron Lett., 1978, 2395–2398.
5, 1471–1474.
23 X-Ray crystallographic data of 20: CCDC 918564, formula =
C30H36Cl6O8 (C28H34O8, 2CHCl3), formula weight = 737.29,
orthorhombic, unit cell parameters: a = 9.7594(4), b =
18.5524(6), c = 18.8335(5), space group = P212121, T =
293 (2) K, Z = 4, R (reflections) = 0.0553 (6815), wR2
(reflections) = 0.1767 (5134).
24 B. S. Siddiqui, M. Rasheed, S. Faizi, Firdous, S. T. Ali,
R. M. Tariq and S. Naqvi, Helv. Chim. Acta, 2003, 86, 3342–
3353.
12 A. Alam, S. Haldar, H. V. Thulasiram, R. Kumar, M. Goyal,
M. S. Iqbal, C. Pal, S. Dey, S. Bindu, S. Sarkar, U. Pal,
N. C. Maiti and U. Bandyopadhyay, J. Biol. Chem., 2012,
287, 24844–24861.
13 E. J. Corey and R. W. Hahl, Tetrahedron Lett., 1989, 30,
3023–3026.
14 A. F. Mateos and A. M. L. Barba, J. Org. Chem., 1995, 60,
3580–3585.
15 S. MacKinnon, T. Durst, J. T. Arnason, C. Angerhofer, 25 K. M. Madyastha and H. V. Thulasiram, J. Agric. Food
J. Pezzuto, P. E. Sanchez Vindas, L. J. Poveda and
M. Gbeassor, J. Nat. Prod., 1997, 60, 336–341.
16 G. E. L. Brandt, M. D. Schmidt, T. E. Prisinzano and
B. S. J. Blagg, J. Med. Chem., 2008, 51, 6495–6502.
Chem., 1999, 47, 1203–1207.
26 P. P. Daramwar, R. Rincy, S. Niloferjahan, R. Krithika,
A. Gulati, A. Yadav, R. Sharma and H. V. Thulasiram, Bio-
resour. Technol., 2012, 115, 70–74.
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