A novel green route for the synthesis of N-phenylacetamides, benzimidazoles and acridinediones using Candida parapsilosis ATCC 7330

Article abstract of DOI:10.1039/c3ra44058c

Biocatalytic preparation of various substituted N-phenylacetamides was carried out for the first time using whole cells of Candida parapsilosis ATCC 7330 under mild reaction conditions with excellent conversions (up to 93%) and good yields (up to 81%). Arylamine N-acetyltransferase (NAT) from whole cells of Candida parapsilosis ATCC 7330 is implicated in the N-acylation which is known to transfer the acetyl group from acetyl CoA (coenzyme A) to aromatic amines. Mechanistic investigation carried out using deuterated ethanol to find the source of acetyl CoA revealed that the reaction proceeds through the formation of acetaldehyde in situ, which is a potential source for the acetyl group of cytoplasmic acetyl CoA. Furthermore, experiments on regioselectivity, carried out with 2-phenylenediamine (2-PDA) resulted in a cyclised product 2-methyl benzimidazole (conversion 99%). The biocatalyst also mediates the cyclization of 3-(phenylamino)cyclohex-2-enones in the presence of the in situ generated acetaldehyde to form acridine-1,8-diones (conversion up to 70%), which is a green process reported here for the first time. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra44058c

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A novel green route for the synthesis of
N-phenylacetamides, benzimidazoles and
Cite this: DOI: 10.1039/c3ra44058c
acridinediones using Candida parapsilosis ATCC 7330†
a
ab
Pula Mahajabeen and Anju Chadha*
Biocatalytic preparation of various substituted N-phenylacetamides was carried out for the first time using
whole cells of Candida parapsilosis ATCC 7330 under mild reaction conditions with excellent conversions
up to 93%) and good yields (up to 81%). Arylamine N-acetyltransferase (NAT) from whole cells of
Candida parapsilosis ATCC 7330 is implicated in the N-acylation which is known to transfer the acetyl
group from acetyl CoA (coenzyme A) to aromatic amines. Mechanistic investigation carried out using
deuterated ethanol to find the source of acetyl CoA revealed that the reaction proceeds through the
formation of acetaldehyde in situ, which is a potential source for the acetyl group of cytoplasmic acetyl
CoA. Furthermore, experiments on regioselectivity, carried out with 2-phenylenediamine (2-PDA)
resulted in a cyclised product 2-methyl benzimidazole (conversion 99%). The biocatalyst also mediates
the cyclization of 3-(phenylamino)cyclohex-2-enones in the presence of the in situ generated
acetaldehyde to form acridine-1,8-diones (conversion up to 70%), which is a green process reported
here for the first time.
(
Received 1st August 2013
Accepted 12th September 2013
DOI: 10.1039/c3ra44058c
www.rsc.org/advances
methods, biocatalysts viz. arylamine N-acetyltransferases (NATs;
EC 2.3.1.5) which catalyze the N-acylation of aromatic amines and
Introduction
N-Acylation of amines is an important and fundamental trans- hydrazines with the acetyl group being transferred from acetyl
1
formation carried out in organic chemistry and amides repre- CoA have been reported from various prokaryotic and eukaryotic
2
10
sent an important class of compounds. Among the various species and the mechanism of these enzymes has been
1
1
chemical methods available for their synthesis, the most studied. Even though it is well known that NATs are involved in
commonly used ones involve the use of reagents, acid chlorides
or acid anhydrides with amines. There are several other methods
such as the modied Staudinger reaction that utilizes azide as
detoxication and bioremediation of soils contaminated with
3
aromatic amines (byproducts of combustion and chemical
12
manufacturing) they have not been extensively used for organic
4
amine
equivalent,
transition-metal
catalyzed
amino- transformations from a synthetic point of view. Hence, there is a
5
6
carbonylation of aryl halides, Beckmann rearrangement, amide constant need to develop an efficient and ‘green’ method for
synthesis using terminal alkynes and the use of the acyl C–H N-acylation of aromatic amines using a biocatalyst i.e. either
7
bond of aldehydes with amines in the presence of a transition- whole cells or enzymes.
8
metal catalyst under oxidative conditions which has been
reported recently. Almost all of these methods use expensive,
moisture sensitive and toxic catalysts which require harsh reac-
tion conditions and protocols which need special handling. One
Herein, we report for the rst time the synthesis of N-phe-
nylacetamides catalysed by the whole cells of C. parapsilosis
ATCC 7330, which has been shown by us earlier to be a versatile
13
biocatalyst which catalyses deracemization,
asymmetric
14
15
more alternative protocol involves direct synthesis of amides reduction and resolution of various aromatic and aliphatic
from alcohols and amines using various ruthenium, rhodium compounds. Various reaction parameters were ne tuned to use
9
and heterogeneous gold catalyst. Apart from the chemical these whole cells in the N-acylation of aromatic amines. The
yeast was also used in the synthesis of 2-methyl benzimidazoles
and acridine-1,8-diones with in situ generated acetaldehyde
Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of from ethanol. Evidently, the enzyme which carries out these
a
Technology Madras, Chennai 600 036, India
b
reactions in the yeast is NAT.
National Centre for Catalysis Research, Indian Institute of Technology Madras,
Chennai 600 036, India. E-mail: anjuc@iitm.ac.in; Fax: +91 44 2257 4102; Tel: +91
4
4 2257 4106
Results and discussion
†
Electronic supplementary information (ESI) available. CCDC 928252. For ESI
Initially, p-methoxy aniline 1b ( p-anisidine) was subjected to
N-acylation using whole cells of C. parapsilosis ATCC 7330 in the
and crystallographic data in CIF or other electronic format see DOI:
0.1039/c3ra44058c
1
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presence of ethanol (5%, v/v) which was used as a cosolvent at
ꢀ
2
5 C. The conversion to N-(4-methoxyphenyl) acetamide 2b was
monitored by HPLC using a reverse phase column at regular
intervals of time. At 24 h maximum conversion (70%) was
observed (Scheme 1). Further increase in the reaction time
decreased the conversion and led to the decomposition of the
product. A similar type of N-acylation was reported with fungi
1
6
17
like Saccharomyces cerevisiae, Candida albicans and Podo-
18
spora anserina which are known to have the enzyme arylamine
N-acetyl transferase (NAT) which acylates various aromatic
amines. To conrm the presence of NAT in C. parapsilosis ATCC
7330 the following experiments were carried out.
N-Acylation of aromatic amines: an insight into the
mechanism
Acetyl CoA is a central metabolite in carbon and energy metab- Fig. 1 Metabolic pathway for acetyl-CoA generation. Enzymes catalysing the
1
9
reactions are as follows: (1) acetyl CoA synthetase; (2) ATP-citrate lyase; (3)
acetaldehyde dehydrogenase; (4) alcohol dehydrogenase; (5) pyruvate decar-
boxylase; (6) pyruvate carboxylase; (7) pyruvate dehydrogenase complex; (8)
citrate synthetase; (9) mitochondrial/peroxisomal carnitine acetyltransferase; (10)
acetyl-carnitine mitochondrial carrier; (11) cytoplasmic carnitine acetyltransfer-
olism and in the biosynthesis of cellular molecules. The two
potential sources for the acetyl group of the cytoplasmic acetyl
20
CoA are either citrate or acetate. Citrate enters the cytoplasm
from the mitochondria to give acetyl CoA via ATP-citrate lyase
(ACL) (Fig. 1), whereas acetyl CoA synthetase (ACS) uses acetate to ase. [Modified from ref. 20–22.]
produce acetyl CoA. The acetate may be obtained from three
sources: externally supplied acetate in the growth medium, from
acetaldehyde by ethanol oxidation mediated by alcohol dehy-
drogenase or from pyruate via pyruvate decarboxylase. Pronk
likely NAT] in C. parapsilosis ATCC 7330 is responsible for
N-acylation. To establish the source of acetyl group in acetyl CoA,
further investigation was carried out using deuterated ethanol.
21
et al. reported that the pyruvate-acetaldehyde-acetate pathway is
the major source of cytoplasmic acetyl CoA in Saccharomyces
22
cerevisiae. Further, Wellen et al. also reported that in Saccharo- Biotransformation of p-anisidine with ethanol-d as cosolvent
6
myces cerevisiae, acetyl CoA was produced outside of mitochon-
When p-anisidine 1b was incubated with whole cells of C. par-
dria from acetate via acetyl CoA synthetase. The yeast used in this
study, Candida parapsilsosis ATCC 7330 also produced acetyl CoA
from acetate which was produced from ethanol added to the cells
as shown by the following observation. When p-anisidine 1b was
incubated in the absence of ethanol only minimal (4%) conver-
sion was observed but in the presence of ethanol, 70% of the
N-acylated product was formed. Conrming that ethanol was the
major source of acetate in acetyl CoA other cosolvents like IPA,
ꢀ
apsilosis ATCC 7330 and (5%, v/v) ethanol-d , at 25 C for 24 h
6
gave the corresponding deuterium labelled acylated product
(
3a, Scheme 2) along with trace amounts of 2b.
1
The H NMR spectrum of the product, showed a drastic
reduction [one-tenth of the regular proton signal of –CH
intensity of the signal corresponding to the methyl group in the
deuterated product (s, at 2.14 ppm, Fig. 2 with EtOH-d ) as
3
] in the
6
compared to the signal corresponding to the methyl group in
N-(4-methoxyphenyl)acetamide (s, 3H at 2.12 ppm, Fig. 2 with
EtOH). The chemical shi values of 2b and 3a are given in
Table 1. These results conrm that ethanol is the source for
acetyl CoA during N-acylation by C. parapsilosis ATCC 7330. In
order to improve the conversion and yield of phenyl acetamides
using whole cells of C. parapsilosis ATCC 7330, various reaction
parameters were optimised.
CH CN and DMSO used for biotransformation gave only trace
3
amounts of the N-acylated product. From this it was clear that the
cosolvent ethanol plays a vital role in the N-acylation of p-anisi-
dine and this warranted further investigation. Two important
control experiments were carried out using (i) only cells
[without the substrate] and (ii) in a separate experiment with
only the substrate [without cells] under identical conditions as
the biotransformation reaction. Both these did not show the
N-acylated product suggesting that an enzymatic process [most
Scheme 1 N-Acylation of aromatic amines using whole cells of Candida para-
psilosis ATCC 7330.
Scheme 2
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1
Fig. 2
H NMR spectrum of N-(4-methoxyphenyl)acetamide and its deuterated
-N-(4-methoxyphenyl)acetamide].
counterpart [2,2,2-d
3
Fig. 3 Effect of pH on N-acylation of p-anisidine by whole cells of Candida
parapsilosis ATCC 7330.
Effect of pH on N-acylation of aromatic amines
The pH dependence of N-acylation using whole cells of C. para- Inhibitor studies on N-acylation of aromatic amines
psilosis ATCC 7330 was determined at different pH values ranging
In an attempt to establish that the N-acylation was mediated by
NAT in C. parapsilosis ATCC 7330 cell free extracts of the yeast
were used. Biotransformation of p-anisidine 1b (0.3 mM in water)
was carried out using the cell free extract of C. parapsilosis ATCC
ꢀ
from 4 to 8.5 at 25 C (Fig. 3) using p-anisidine 1b as substrate.
The maximum conversion (69%) to N-(4-methoxyphenyl) acet-
amide 2b was observed at pH 7. Further increase of pH to 8.5
decreased the conversion to 31%. However, the conversion was
7
330 (5 mL) in the absence of cosolvent ethanol and incubated
almost identical in water (70%) and 10 mM potassium phosphate
buffer of pH 7 (69%) hence, water was used instead of buffer for
further reactions. Reportedly, puried Saccharomyces cerevisiae
NAT and Candida albicans NAT showed maximum activity at pH 9
ꢀ
for 6 h at 35 C. Notably, even in the absence of ethanol as
cosolvent, maximum conversion (72%) of N-(4-methoxyphenyl)-
acetamide 2b was observed. This indicates that in the cell free
extract, acetyl CoA is readily available for N-acylation of a
minimum amount of substrate (0.3 mM). But in the case of whole
cells, where higher concentrations of substrate (39 mM) was
used, acetate is required for the acetyl CoA which comes from the
1
6,17
and 8 respectively.
Effect of temperature on N-acylation of aromatic amines
The temperature dependence of C. parapsilosis ATCC 7330 added ethanol.
during N-acylation was studied by determining the conver-
Further, inhibitor studies were carried to show the involve-
sion of p-anisidine 1b to the N-(4-methoxyphenyl)acetamide ment of NAT in N-acylation of aromatic amines. The cell free
2
b using whole cells at different temperatures ranging extract of C. parapsilosis ATCC 7330 was preincubated for
from 20–45 C (Fig. 4). The maximum conversion (93%) to 30 min at 35 C, with different concentrations (0.25, 0.5, 1 and 2
ꢀ
ꢀ
ꢀ
N-(4-methoxyphenyl) acetamide 2b was observed at 35
C
mM) of iodoacetamide, followed by the subsequent addition of
ꢀ
which did not change at 40 C but further increase in p-anisidine (0.3 mM). Aer 6 h a drastic reduction in the
temperature to 45 C decreased the conversion to 44%. The conversion (38, 24, 10 and 6%) with the increased concentration
cell biomass was more at 35 C compared to 45 C. Hence, of iodoacetamide was observed. Similar type of dose-dependent
further reactions were carried out at 35 C. Maximum NAT inhibition was observed by Fang et al. with iodoacetamide as
ꢀ
ꢀ
ꢀ
ꢀ
17
1
6,17,23
activity shown by various species
ture range of 30–40 C.
was in the tempera- an inhibitor for Candida albicans NAT. When the cell free extract
ꢀ
2+
was preincubated with Zn ions at different concentrations
Table 1 Chemical shift (d) values of 2b and 3a
d
H
(ppm)
d
C
(ppm)
d
H
(ppm)
C
d (ppm)
2
3
6
7
7
.12 (3H, s, H
.77 (3H, s, H
.81–6.84 (2H, m, H
.36–7.39 (2H, m, H
.52 (1H, br s, NH)
1
)
)
24.3 (C
55.4 (C
1
)
)
23.5 (C
55.4 (C
1
7
, m)
)
7
7
7
3.78 (3H, s, H )
5
)
)
114.1 (2 ꢁ C
5
4
)
)
6.83–6.86 (2H, m, H
7.25 (1H, br s, NH)
7.36–7.39 (2H, m, H
5
)
)
114.1 (2 ꢁ C
5
)
)
4
122.0 (2 ꢁ C
121.9 (2 ꢁ C
4
131.0 (C
3
)
6
)
2
)
4
130.9 (C
156.4 (C
168.2 (C
3
6
2
)
)
)
1
1
56.4 (C
68.4 (C
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probably due to steric reasons and 1e with a strong electron
withdrawing nitro group in the para position was not acylated,
possibly due to the unavailability of the lone pair of electrons on
the nitrogen atom of the amino group.
The question of regioselectivity: biotransformation of
diamines
In order to check the regioselectivity of the biocatalyst towards
Fig. 4 Effect of temperature on N-acylation of p-anisidine by whole cells of N-acylation, 2-phenylenediamine (2-PDA) was used as a model
Candida parapsilosis ATCC 7330.
substrate. When 2-PDA 1g in (5%, v/v) ethanol was incubated
with whole cells of C. parapsilosis ATCC 7330 at 35 C, the
ꢀ
expected products i.e. monoacylated 2-aminoacetanilide [Aoki
0
.01, 0.1, 1 and 2 mM as mentioned above, the conversion
10a,24
et al.
using whole cells of Bacillus cereus strain] or diacylated
dropped to 88, 39, 29 and 8% respectively, suggesting that NAT
could be involved in the N-acylation reaction. Similarly, Lee
et al. also reported that divalent cations such as Cu and Zn
completely abolished the NAT activity of Saccharomyces cer-
evisiae at a concentration >0.1 mM.
0
1,2-diacetamidobenzene
(N,N -1,2-phenylenebis-acetamide)
were not observed. Instead, a cyclised product 2-methyl-
benzimidazole 2g was formed as a sole product in 99%
conversion and 74% yield in 10 h (Scheme 4). The conversion to
16
2+
2+
2
g was monitored by HPLC using a reverse phase column at
regular intervals of time and at 10 h the maximum conversion
99%) was observed. Control experiments done in parallel
(
Effect of substitution on aromatic ring during acylation
without the whole cells under identical conditions showed no
product. Further, an experiment with only acetaldehyde (10%,
v/v) in presence of water when added to 1g resulted in the
formation of 2g indicating that the reaction proceeds through
the formation of acetaldehyde. To conrm the same, when
Under the optimized reaction conditions, various aromatic
substituted amines were acylated upon incubation with whole cells
of C. parapsilosis ATCC 7330 at 35 C for 24 h as shown in (Scheme
ꢀ
1, Table 2). p-Methoxy aniline 1b gave N-(4-methoxyphenyl)acet-
amide 2b in excellent conversion (93%) compared to p-methyl 1c
and p-chloro 1d anilines respectively, which gave their corre-
sponding N-phenyl acetamides 2c (85%) and 2d (80%) in good
conversion. Unsubstituted aniline 1a gave phenylacetamide 2a in 13
moderate conversion (65%). The conversion to N-acetamides with
o-bromo 2f (13%) and 1-naphthyl (8%) substitutions was very low
2
7
-PDA was incubated with whole cells of C. parapsilosis ATCC
ꢀ
330 and (5%, v/v) ethanol-d
6
, for 10 h at 25 C as mentioned
1
above and the product obtained was characterized by H and
1
C NMR spectroscopy, the H NMR spectrum of the puried
compound 2-(methyl-d )-1H-benzimidazole, (Fig. 5, with EtOH-
3
6
d ) showed disappearance of the signal corresponding to the
methyl group in 2-methyl-benzimidazole (s, 3H at 2.61 ppm,
Fig. 5, with EtOH). The chemical shi values of 2g and 3b are
given in Table 3. This conrmed that the acetaldehyde
produced during the biotransformation was involved in cycli-
Table 2 Biocatalytic preparation of various N-phenylacetamides using the
whole cells of Candida parapsilosis ATCC 7330
a
b
S. no.
1
R
Product
Conversion (%)
Yield (%) sation to give 2-d -methyl benzimidazole (Scheme 3).
3
Benzimidazole and its derivatives have found applications in
various therapeutic areas as anti-cancer, anti-tumor, anti-
H 1a
65
93
56
81
25
bacterial, anti-fungal, anti-viral agents and have an important
role in chemistry as ligands of transition metals for modeling
26
biological systems. Among the classical methods of benz-
imidazole synthesis one needs the coupling of 2-PDA with
2
3
3
p-OCH 1b
27
carboxylic acids or their derivatives which requires high
temperatures and strong acidic conditions. The other method
involves condensation of 2-PDA with aldehyde to give aniline
Schiff's bases which are involved in oxidative cyclo-dehydroge-
p-CH
3
1c
85
79
28
nation. Though the temperature required here is low there is
the disadvantage of using stoichiometric amounts of oxidizing
4
5
p-Cl 1d
p-NO 1e
80
74
2
Nd 2e
Nd
—
6
o-Br 1f
13
—
a
The conversion to the product was monitored by HPLC using C-18
b
column. Isolated yield. Nd: not determined.
Scheme 3
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Scheme 4 Synthesis of some benzimidazoles from whole cells of Candida par-
apsilosis ATCC 7330.
1
Fig. 5
3
H NMR spectrum of 2-methyl-benzimidazole and 2-(methyl-d )-1H-
benzimidazole.
Table 4 Biocatalytic preparation of benzimidazoles 2g- 2h using the whole cells
of Candida parapsilosis ATCC 7330
agents such as DDQ, nitrobenzene (high boiling point), 1,4-
benzoquinone, tetracyanoethylene, benzofuroxan, Pb(OAc)
MnO , NaHSO and Na . Herein, we report for the rst time
a
b
S. no.
1
R
Product
Conversion (%)
Yield (%)
4
,
2
3
2 2 5
S O
a ‘green’ synthesis of benzimidazoles using mild reaction
conditions and the biocatalyst, C. parapsilosis ATCC 7330.
To generalise the reaction, when various diamines (Scheme 4,
Table 4) were incubated with whole cells of C. parapsilosis
H 1g
99
79
84
65
2
3
3-CH 1h
ꢀ
ATCC 7330 at 35 C for 10 h, simple 2-phenylenediamine 1g gave
2-methylbenzimidazole 2g with 99% conversion and 84% yield
whereas a 3-CH 1h and 4-Cl 1i substitution on 2-PDA gave its
3
3
4-Cl 1i
73
61
corresponding imidazoles 2h and 2i with 79% and 73% conver-
sion and 65% and 61% yields respectively.
a
Various cyclised products can be formed by utilizing the
acetaldehyde generated during the biotransformation with
whole cells of C. parapsilosis ATCC 7330. The cyclised product
formed in case of 2-PDA 1g encouraged us to check the forma-
tion of acridine-1,8-diones with whole cells of C. parapsilosis
ATCC 7330 taking forward the ‘green’ and easier approach
using acetaldehyde obtained during the biotransformation.
The conversion to the product was monitored by HPLC using C-18
column. Isolated yield.
b
of acridine-1,8-diones using 3-(phenylamino)cyclohex-2-enones
and aliphatic aldehydes like acetaldehyde at room temperature.
Applications of various substituted acridine-1,8-diones
include, anti-microbial, anti-malarial and anti-cancer activi-
31
ties. These molecules also exhibit laser activity, are used as
Biocatalytic preparation of N-substituted acridine-1,8-diones
by in situ generated acetaldehyde
32
photo initiators and in the production of dyes.
Various phenyl substituted 3-(phenylamino) cyclohex-2-
29
Among the various methods available for the synthesis of enones were used as substrates for the synthesis of acridine-1,8-
acridine-1,8-diones, a few methods which use enaminones as diones. Initially, 3-(phenylamino)cyclohex-2-enone 4a (0.02
starting materials involves the condensation of enaminones mmol, 4 mg) dissolved in ethanol (100 mL) when incubated with
(
formed from 1,3-cyclohexanedione derivatives) and an whole cells of C. parapsilosis ATCC 7330 (1.6 g) as a standard
ꢀ
aromatic aldehyde under reux conditions using water as substrate on an orbital shaker at 200 rpm at 35 C for 24 h, gave
30
solvent or use of P O in isopropanol or use ionic liquids. We 9-methyl-10-phenyl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-
2
5
observed that in literature there are no reports for the synthesis dione 5a with a maximum conversion of 40% (Scheme 5). The
Table 3 Chemical shift (d) values of 2g and 3b
d
H
(ppm)
d
C
(ppm)
d
H
(ppm)
d
C
(ppm)
2
7
7
7
.61 (3H, s, H
1
)
14.6 (C
1
)
—
14.1 (C
1
, m)
.19–7.22 (2H, m, H
.51–7.55 (2H, m, H
.86 (br s, NH)
5
)
)
114.3 (2 ꢁ C
122.2 (2 ꢁ C
138.3 (2 ꢁ C
4
5
3
)
)
)
7.19–7.23 (2H, m, H
7.52–7.56 (2H, m, H
5
)
)
114.4 (2 ꢁ C
122.2 (2 ꢁ C
138.5 (2 ꢁ C
4
5
3
)
)
)
4
4
151.2 (C
2
)
2
151.1 (C )
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chloro 5d (48%) substitutions show no improvement in
conversion. In the case of a strong electron withdrawing group
like nitro in the para position, the product was not observed
even aer increasing the reaction time to 48 h. This clearly
indicates that the conversion and yield of acridine-1,8-diones
depends on the availability of lone pair of electrons on nitrogen
atom of amino group.
Conclusion
Herein, we report for the rst time, the N-acylation of various
aromatic amines to N-phenyl acetamides using whole cells of
Candida parapsilosis ATCC 7330 with excellent conversions (up
to 93%) and yields (up to 81%). The mechanistic investigation
carried out using deuterated ethanol as cosolvent showed the
involvement of in situ generated acetaldehyde from deuterated
Scheme 5 Preparation of N-substituted acridine-1,8-diones catalyzed by whole
cells of Candida parapsilosis ATCC 7330.
formation of the product was monitored by HPLC using a
reverse phase column at regular intervals of time. To improve
the conversion further, a fresh biomass (1.6 g) along with the
cosolvent ethanol (100 mL) was added together at the end of
ꢀ
ethanol. Various reaction parameters like temperature (35 C)
and pH (7) were optimised for N-acylation of p-anisidine to get
N-(4-methoxyphenyl) acetamide in higher conversion (93%) and
yield (81%) using whole cells of Candida parapsilosis ATCC
24 h. Aer 48 h of reaction time the conversion [formation of 5a
increased to 60%. Hence, further reactions were continued by
adding fresh biomass and ethanol at the end of 24 h. The
7
330. Biotransformation of 2-phenylenediamine gave a cyclised
product 2-methyl benzimidazole with excellent conversion
99%) and yield (84%) instead of the mono- or diacylated
1
13
formation of 5a was conrmed by H, C NMR, FT-IR spectra.
The structure of 5a was further conrmed by X-ray crystallog-
raphy (Fig. 6). As shown in Table 5 various substituents on the
phenyl ring of acridinediones were introduced to study the
electronic and steric effects. A strong electron donating group
like methoxy substitution in para position of phenyl ring 5b
increases the conversion to 70%, whereas methyl 5c (51%) and
(
products. A novel, green bio-based strategy for the preparation
of acridine-1,8-diones with good conversion (up to 70%) and
yield (up to 61%) was developed for the rst time using the
biocatalyst Candida parapsilosis ATCC 7330.
Experimental
General method
Candida parapsilosis ATCC 7330 was purchased from ATCC,
Manassas, VA 20108, USA. All chemicals used for media prepa-
ration and all substrates were purchased locally. Ethanol-d
6
1
(
anhydrous, 99.5%) was purchased from Sigma Aldrich. H and
1
3
C NMR spectra were recorded in CDCl
AVANCE III 500 MHz spectrometer operating at 500 MHz and
25 MHz. Chemical shis are expressed in ppm values using
3
solution on a Bruker
1
TMS as an internal standard. Infrared spectra were recorded on a
Shimadzu IR 470 Instrument. HPLC analysis was done on a Jasco
PU-1580 liquid chromatograph equipped with a manual injector
(20 mL) and a PDA detector. The conversion of the N-phenyl
acetamides, 2-methyl benzimidazoles and acridine-1,8-diones
was determined by reverse phase C18 column using acetonitrile–
water (40 : 60) solvent mixture with ow rate 0.5 mL min . TLC
ꢂ1
Fig. 6 ORTEP depiction of compound 5a.
was carried out using Kieselgel 60 F254 aluminium sheets (Merck
1
.05554). X-ray crystallographic analysis was performed with a
Table 5 Biocatalytic preparation of various acridine-1,8-diones using the whole
cells of Candida parapsilosis ATCC 7330
Bruker AXS Kappa APEX II single crystal CCD Diffractometer
equipped with graphite-monochromated MoKa radiation (l ¼
˚
a
b
0.71073 A) at room temperature.
Entry
R
Conversion (%)
Yield (%)
5
5
5
5
5
a
b
c
d
e
H
p-OCH
p-CH
p-Cl
p-NO
60
70
51
48
Nd
48
51
45
36
—
Culture medium used for the growth of C. parapsilosis ATCC
3
33
7330
3
Candida parapsilosis ATCC 7330 was grown in yeast malt broth
medium (50 mL) in 250 mL Erlenmeyer asks incubated at
2
ꢀ
a
25 C, 200 rpm. The cells were harvested by centrifuging the 40 h
The conversion to the product was monitored by HPLC using C-18
b
ꢀ
column. Isolated yield. Nd: not determined.
culture broth at 10 000 rpm for 10 min at 4 C and subsequently
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washed with distilled water. The process was repeated twice, and 9-Methyl-10-(4-methoxyphenyl)-3,4,6,7,9,10-hexahydroacridine-
the wet cells were used for biotransformation.
1,8(2H,5H)-dione (5b)
ꢀ
1
Yellow colour solid, mp 181–183 C H NMR (500 MHz, CDCl
3
) d
), 2.21–2.27 (m,
), 3.86 (s, 3H), 4.15 (q, 1H, J ¼
Preparation of the cell free extract by cell disruption using a
sonicator
1
.00 (d, 3H, J ¼ 6.5 Hz), 1.76–2.09 (m, 8H, 4CH
H, CH ), 2.38–2.43 (m, 2H, CH
2
34
2
2
2
Potassium phosphate buffer (10 mM) of pH 7.0 was used to 6.5, C
9
–CH), 6.97 (d, 2H, J ¼ 8.5 Ar-H), 7.1 (d, 2H, J ¼ 8.0 Ar-H).
1
3
resuspend the harvested cells (32 g of wet cells) of C. parapsilosis
C NMR (125 MHz, CDCl
3
) d 21.3, 21.5, 22.8, 28.1, 36.8, 55.6,
ꢂ1
ATCC 7330 (50 mL of lysis buffer, 2 mM of PMSF). The 114.9, 116.9, 130.3, 131.7, 152.2, 159.7, 196.5. IR (KBr, cm
)
suspension was then subjected to cell disruption by ultra- 3430, 3260, 2959, 2925, 2886, 1637, 1573, 1508, 1373, 1278,1232,
sonicator (a pulse of 1 s on and 2 s off, for 5 min, 40% ampli- 1183, 1129, 1032, 949. HRMS: calcd for C21H24NO 338.1882
3
tude). The undisrupted cells and debris were pelleted by [M + 1]; obs. 338.1880.
ꢀ
centrifuging the homogenate at 10 000ꢁg for 15 min at 4 C.
The supernatant was used as the cell free extract for further 9-Methyl-10-(4-methylphenyl)-3,4,6,7,9,10-hexahydroacridine-
ꢂ1
experiments. Protein concentration (2.4 mg mL ) in the cell 1,8(2H,5H)-dione (5c)
free extract was estimated by the Bradford method.
1
ꢀ
Brown colour solid, mp 191–193 C H NMR (500 MHz, CDCl ) d
3
1
.00 (d, 3H, 6.5 Hz), 1.78–2.11 (m, 8H, 4CH
CH ), 2.37–2.41 (m, 2H, CH
), 2.42 (s, 3H), 4.15 (q, 1H, J ¼ 6.5,
–CH), 7.07 (d, 2H, J ¼ 8.0 Ar-H), 7.28 (d, 2H, J ¼ 8.0 Ar-H).
) d 21.1, 21.3, 21.6, 22.9, 28.1, 36.8, 116.9,
2
), 2.21–2.27 (m, 2H,
Biotransormation of p-anisidine using cell free extract of C.
parapsilosis ATCC 7330
2
2
13
C
9
C
To 5 mL of cell free extract, p-anisidine (0.3 mM) was added and NMR (125 MHz, CDCl
the reaction mixture was incubated at 35 C for 6 h at 200 rpm. 129.3, 130.6, 136.6, 139.3, 152.0, 196.5. IR (KBr, cm ) 3427,
3
ꢂ1
ꢀ
Aer the formation of the product the reaction mixture was 3261, 2958, 2923, 2868, 1722, 1635, 1572, 1511, 1375, 1285,
extracted using ethyl acetate and the organic layer was dried 1237, 1188, 1130, 954. HRMS: calcd for C21
over anhydrous sodium sulfate. The solvent was removed by [M + 1]; obs. 322.1794.
rotary evaporator. All the experiments were done in triplicate
H24NO 322.11807
2
and the control experiment which was done with heat treated 9-Methyl-10-(4-chlorophenyl)-3,4,6,7,9,10-hexahydroacridine-
cell free extract showed no product.
1,8(2H,5H)-dione (5d)
ꢀ
1
Pale yellow solid, mp 187–189 C H NMR (500 MHz, CDCl ) d
3
Typical procedure for N-acylation of aromatic amines using
whole cells of C. parapsilosis ATCC 7330
1
.01 (d, 3H, 6.5 Hz), 1.80–1.96 (m, 6H, 3CH ), 2.05–2.11 (m, 2H,
2
2 2 2
CH ), 2.22–2.29 (m, 2H, CH ), 2.39–2.45 (m, 2H, CH ), 4.15 (q,
The substrate, p-anisidine (0.58 mmol, 72 mg) dissolved in 1H, J ¼ 6.5, C –CH), 7.16 (d, 2H, J ¼ 8.5 Ar-H), 7.48 (d, 2H, J ¼ 9.0
9
1
3
ethanol (1.8 mL) was added into a 150 mL Erlenmeyer ask Ar-H). C NMR (125 MHz, CDCl
3
) d 21.3, 21.6, 22.9, 28.1, 36.7,
ꢂ1
containing the cells (18 g wet wt) in water (15 mL) and stirred on 117.3, 131.0, 135.2, 136.9, 137.8, 151.1, 196.3. IR (KBr, cm
)
ꢀ
an orbital shaker at 200 rpm for 24 h at 35 C. The reaction was 3435, 2959, 2927, 2873, 2857, 1726, 1633, 1570, 1383, 1271,
performed in triplicates. The conversions were determined by 1186, 1119. HRMS: calcd for C20
HPLC using a C18 column with ow rate of 0.5 mL min using 342.1255.
H21NO
2
Cl 342.1261 [M + 1]; obs.
ꢂ1
acetonitrile–water (40 : 60) as eluent. Aer the completion of
reaction, the reaction mass was extracted with ethylacetate (3 ꢁ d3-Methyl-N-(4-methoxyphenyl)acetamide (3a)
0 mL) and dried over anhydrous Na SO . The crude product
was puried by column chromatography on silica gel (mesh size
00–200) using ethyl acetate and hexane mixture (25 : 75), to
1
2
4
1
H NMR (500 MHz, CDCl
3
) d 3.78 (s, 3H), 6.83–6.86 (m, 2H), 7.25
13
(1H, br s, NH), 7.36–7.39 (m, 2H). C NMR (125 MHz, CDCl ) d
3
1
2
3.5, 55.4, 114.1, 121.9, 130.9, 156.4, 168.2.
give the 2b in 54% yield. Similar procedure was followed for the
N-acylation of the other amines. Under identical conditions
control experiments were done without whole cells. The spectral
data of new compounds are given below.
2
-d
3
-Methyl-benzimidazole (3b)
) d 7.19–7.23 (m, 2H), 7.52–7.56 (m,
1
H NMR (500 MHz, CDCl
3
1
3
2
1
H). C NMR (125 MHz, CDCl
51.1.
3
) d 14.1, 114.4, 122.1, 138.5,
9
-Methyl-10-phenyl-3,4,6,7,9,10-hexahydroacridine 1,8(2H,5H)-
dione (5a)
Yellow colour solid, mp 179–180 C H NMR (500 MHz, CDCl
ꢀ
1
X-ray analysis of compound 5a
), 2.25–2.43 (m, Empirical formula ¼ C40
3
) d
1
4
7
3
3
1
3
.02 (d, 3H, J ¼ 7 Hz), 1.85–1.95 (m, 8H, 4CH
2
H
42
N
2
O
4
, formula weight ¼ 614.76,
˚
H, 2CH
2
), 4.17 (q, 1H, J ¼ 6.5, C
9
–CH), 7.19–7.21 (m, 2H), 7.45– temperature ¼ 293(2) K, wavelength ¼ 0.71073 A, crystal system,
1
3
.51 (m, 3H). C NMR (125 MHz, CDCl ) d 21.3, 21.5, 22.8, 28.1, space group monoclinic, P2 /c, unit cell dimensions a ¼
3
1
ꢂ1
˚
˚
6.8, 117.0, 129.1, 129.7, 139.2, 151.7, 196.4. IR (KBr, cm
)
11.639(5) A alpha ¼ 90.000(5) deg., b ¼ 13.976(5) A beta ¼
˚
422, 3259, 3052, 2948, 2920, 2866, 1634, 1590, 1567, 1494, 104.729(5) deg., c ¼ 20.968(5) A gamma ¼ 90.000(5) deg.,
3
ꢂ3
˚
375, 1288, 1236, 1185, 1132, 956. HRMS: calc. for C20
H
22NO
2
volume ¼ 3299(2) A Z, calculated density ¼ 4, 1.238 mg m
,
ꢂ1
08.1651 [M + 1]; obs. 308.1636.
absorption coefficient ¼ 0.079 mm , F(000) ¼ 1312, crystal size
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¼
0.30 ꢁ 0.20 ꢁ 0.20 mm, theta range for data collection ¼ 1.77
to 25.00 deg., limiting indices ꢂ13 # h # 13, ꢂ16 # k # 16, ꢂ24
l # 24, reections collected/unique ¼ 31 490/5804 [R
Z. Wang, Angew. Chem., Int. Ed., 2011, 50, 8917–8921; (e)
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2010, 122, 404–408; Angew. Chem., Int. Ed., 2010, 49, 394–398.
#
¼
(
int)
0
.0354], completeness to theta ¼ 25.00100.0%, absorption 10 (a) S. Mulyono, Y. Takenaka, S. Sasano, Murakami and
correction ¼ semi-empirical from equivalents, max. and min.
transmission ¼ 0.982 and 0.931, renement method ¼ full-
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