Synthesis of γ-nitroaldehydes containing quaternary carbon in the α-position using a 4-oxalocrotonate tautomerase whole-cell biocatalyst

Article abstract of DOI:10.1039/c4ra05517a

Synthesis of γ-nitroaldehydes from branched chain aldehydes and a range of α,β-unsaturated nitroalkenes was achieved by a whole-cell biocatalytic reaction using 4-oxalocrotonate tautomerase as catalyst. Under mild conditions, cyclic and acyclic branched a

Full text of DOI:10.1039/c4ra05517a

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Synthesis of g-nitroaldehydes containing
quaternary carbon in the a-position using a 4-
oxalocrotonate tautomerase whole-cell
biocatalyst†
Cite this: RSC Adv., 2014, 4, 60502
Jelena Radivojevic,ab Gordana Minovska,b Lidija Senerovic,b Kevin O'Connor,c
Predrag Jovanovic,d Vladimir Savic,d Zorana Tokic-Vujosevic,d Jasmina Nikodinovic-
b
a
*
*
Runic and Veselin Maslak
Synthesis of g-nitroaldehydes from branched chain aldehydes and a range of a,b-unsaturated nitroalkenes
was achieved by a whole-cell biocatalytic reaction using 4-oxalocrotonate tautomerase as catalyst. Under
mild conditions, cyclic and acyclic branched aldehydes were converted into synthetically valuable
quaternary carbon containing g-nitroaldehydes. The yields of the desired products were influenced by
reaction condition parameters such as organic solvent, temperature and pH. The whole-cell biocatalytic
approach to the generation of a,a-substituted g-nitroaldehydes was compared to the organocatalytic
approach involving the lithium salt of phenylalanine as a catalyst. As the resulting g-nitroaldehydes
exhibited moderate antifungal activity and mild in vitro cytotoxicity against human fibroblasts (0.2–0.4
mM) they could further be examined as potentially useful pharmaceutical synthons.
Received 9th June 2014
Accepted 4th November 2014
DOI: 10.1039/c4ra05517a
www.rsc.org/advances
useful g-nitroaldehydes (Scheme 1). g-Nitroaldehydes are
precursors for g-aminobutyric acid analogs (GABAs) exhibiting
Introduction
a range of pharmacological activities including antidepressant,
anticonvulsant, anxiolytic and others.10,11 There has been
increasing interest in the synthesis of new GABA derivatives
which can be potent drugs in the treatment of neurodegenera-
tive disorders.12 Furthermore, the presence of nitro group allows
for further transformations to valuable functionalities such as
amines.13–15
g-Nitroaldehydes can be obtained by two different Michael-
type additions: (i) from addition of aldehydes and a,b-unsatu-
rated nitroalkenes and (ii) from addition of nitroalkanes and
a,b-unsaturated aldehydes (Scheme 1a). However, in this
Synthetic methods allowing carbon–carbon bond formation
under mild conditions are of great interest, especially when they
address the problem of stereogenic quaternary carbon forma-
tion.1,2 Quaternary carbon stereocenters (carbon centers con-
taining four different non-hydrogen substituents) are present in
a wide variety of organic substances, including bioactive natural
products and medicinal agents.3,4 Nevertheless, their genera-
tion still remains a challenge. Michael-type reactions are one of
the most utilised reactions for the formation of C–C bonds in
general, and they also afford the possibility of quaternary center
introduction when a,a-disubstituted aldehydes are employed.5–8
Furthermore, nitrostyrenes as highly reactive Michael acceptors
are widely utilized in these type of reactions.9 Michael-type
addition is an effective method for obtaining synthetically
aFaculty of Chemistry, University of Belgrade, Studentski Trg 12-16, 11000 Belgrade,
Serbia. E-mail: vmaslak@chem.bg.ac.rs; Fax: +381-11-218-4330; Tel: +381-11-333-
3655
bInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade,
Vojvode Stepe 444a, P.O. Box 23, 11010 Belgrade, Serbia. E-mail: jasmina.
nikodinovic@gmail.com; Fax: +381-11-397-5808; Tel: +381-11-397-6034
cSchool of Biomolecular and Biomedical Sciences, Centre for Synthesis and Chemical
Biology, University College Dublin, Beleld, Dublin 4, Ireland
dDepartment of Organic Chemistry, Faculty of Pharmacy, University of Belgrade,
Vojvode Stepe 450, 11221 Belgrade, Serbia
† Electronic supplementary information (ESI) available: Copies of the 1H NMR
and 13C NMR spectra; supplementary Table 1. See DOI: 10.1039/c4ra05517a
Scheme
carbon in the a-position.
1 Michael-type additions for introduction of quaternary
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system the only way to introduce quaternary centre in the a-
position of nitroaldehyde is the reaction of branched aldehyde
with a nitroalkene (Scheme 1b). Michael-type additions of
unmodied aldehydes to nitroolens were successfully devel-
oped through organocatalytic approaches.16,17 They usually
involve reactions of primary activated amines,15,18–20 secondary
amines,19,21 alkaline metal salts of primary amino acids,22,23
peptides24 or functionalized chiral ionic liquids.25 Although
organocatalytic methods are versatile and valuable,26 they are in
this case dependant on application of excessive amounts of
proline or peptide based catalysts (10–20% mol%)24 and on the
presence of usually chlorinated organic solvents, making their
environmental impact high.
Only recently, due to the discovery of the versatile and
promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT)
from Pseudomonas putida mt-2, which contains a terminal
proline (Pro1) residue, Michael-type additions of aldehydes to
nitroolens were described.27–29 It was proposed that the most
likely catalytic mechanism of the 4-OT-catalyzed Michael-type
additions also depend on proline-based catalysis and involves
the formation of a nucleophilic enamine intermediate, which
in turn reacts with the double bond of the nitro olen creating
a new C–C bond aer which the product is released from 4-
OT's Pro1 by hydrolysis.27 Miao and co-authors have described
that this particular enzyme in the puried form accepts in
addition to acetaldehyde, a range of linear aldehydes up to
octanal in the reaction with trans-nitrostyrene.28 During the
same study, they found that this system would not accept
branched aldehydes including isobutanal nor acetone as
donors.
Previously we have described a biocatalytic process intensi-
cation utilising whole-cell biocatalyst based on a 4-OT enzyme
(Escherichia coli BL21 (4-OT)) originating from Pseudomonas
putida mt-2 strain, in the biocatalytic synthesis of 4-nitro-3-aryl-
butanals.30 Here we report on the extension of our biocatalytic
strategy by showing that, 4-OT whole-cell biocatalyst also
accepts variety of substituted aromatic and heterocyclic nitro-
alkenes as acceptors and branched aldehydes namely iso-
butanal, 2-ethylbutanal, cyclohexanecarbaldehyde and 2-
methylpentanal as donors for the Michael-type addition
(Scheme 2; Fig. 1A). In addition, we examined the antimicrobial
and cytotoxic effects of resulting g-nitroaldehydes (Fig. 1B). All
products generated biocatalytically were also generated using
asymmetric organocatalytic synthesis involving lithium salt of
phenylalanine22,23 allowing for the direct comparison of the two
methods.
Fig. 1 Structures of (A) branched donors and (B) g-nitroaldehyde
products of Michael-type additions catalyzed by 4-OT.
Results and discussion
Biocatalytic synthesis of 2,2-dimethyl-4-nitro-3-phenylbutanal
(1a) and inuence of reaction conditions
We started our investigations by exploring the reaction of b-
nitrostyrene (1) with isobutanal (a) using bacterial cells
expressing 4-OT. A preparative scale reaction was performed
with 1 (2 mM) and a (20 mM) in 20 mM NaH2PO4 buffer (pH 7.2)
at 28 ꢀC (Scheme 2). Due to the poor solubility of nitrostyrene in
NaH2PO4 buffer, it was supplied from an ethanol stock solution
(200 mM) so the reaction contained 1% (v/v) EtOH. As expected,
upon completion (ꢁ4 h) 2,2-dimethyl-4-nitro-3-phenylbutanal
(1a) was formed as a result of Michael-type addition of 1 and
a. Surprisingly, signicant amount of 4-nitro-3-phenylbutanal
(1a*), a product of Michael-type addition of acetaldehyde to 1,
was also isolated. The respective yields were 26 and 15% (Table
1, Entry I). In the control reaction with no microbial cells or
autoclaved cells that should contain no enzymatic activity, no
reaction occurred, while in the reaction of cells containing
expression plasmid without the gene encoding the 4-OT
enzyme, the reduction of 1 to 2-phenyl-nitroethane (5–10%
yield) was observed regardless of the presence of aldehyde due
to the presence of non-specic reductases in the host E. coli
BL21 cells.31 2-Phenyl-nitroethane was not detected during
biotransformations with E. coli BL21 (4-OT).
The addition of 1 (neat substance) to the aqueous reaction
resulted in poor solubility and availability of the substrate,
Scheme 2 Tautomerase based whole-cell catalyzed Michael addition of isobutanal to b-nitrostyrene.
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Table 1 Synthesis of 1a (2,2-dimethyl-4-nitro-3-phenylbutanal) through biotransformation of b-nitrostyrene (1) and isobutanal (a) by
recombinant E. coli BL21 (4-OT) using 5 g CDW Lꢂ1 at 28 ꢀC, pH 7.2 in 60 mL volume
Reaction time
(h)
1 Depletion ratea (mmol per min per g
CDW)
Entry
Solvent for 1
Product
Yield (%)
I
Ethanol
Isopropanol
2-Butanol
DMSO
1a and 1a*
4
4
4
6
20
1.8
1.7
1.7
1.2
0.21
26 and 15
II
III
IV
V
1a
1a
1a
1a
54
60
28
10
No
a
Standard errors were between 1 and 3%.
coinciding with a low substrate depletion rate and low yield of nitrostyrene which caused an increase in product yield and
1a (10% aer 20 h incubation) (Table 1; Entry V). Furthermore, reduced the amount of generated side products.28 In the current
no 1a* was formed, suggesting that the presence of EtOH was study lowering the pH of reaction buffer from 7.2 to 5.5 resulted
crucial for the formation of 1a*. Thus we examined the inu- in a considerably lower product yield (26% instead 60%). The
ence of solvent used for the preparation of nitrostyrene stock observed reduction of the product yield to 26% and 15% (ESI
solution on product formation and yield (Table 1). When iso- Table 1;† Entry II and IV) could be explained by the poorer
propanol and 2-butanol were used for preparation of stock transfer rates across the membrane of the substrates under these
solution of 1, overall yields of 1a were 54 and 60%, respectively reaction conditions. Control reaction using cell-free extracts of
(Table 1; Entry II and III). When DMSO was used, product yield the E. coli BL21 (4-OT) as catalyst required longer time for the
was approximately half of that obtained compared to when Michael adduct to be formed, but decrease of the product yield
isopropanol and 2-butanol were used (Table 1; Entry IV). It is was not observed (ESI Table 1;† Entry II0–IV0). This may be due to
possible that an alcohol inducible dehydrogenase/acetaldehyde the fact that lowering the pH of the buffer in the case of whole-
reductase was present in E. coli BL21 host cells and due to its cells did not change the pH inside the cells, where the enzyme
activity, a portion of EtOH present in the reaction was converted is located, while it negatively affected substrate uptake or trans-
to acetaldehyde that was further added to b-nitrostyrene by the port. In the case of both puried enzyme28 and whole-cell bio-
activity of 4-OT resulting in formation of 1a* (Table 1; Entry I). catalyst, lowering the pH of the reaction buffer did not affect the
Indeed, numerous alcohol dehydrogenases including members enantioselectivity (ESI Table 1;† Entry II).
of both short-chain and medium-chain dehydrogenase/
As no improvement of stereoselectivity was observed when
reductases enzyme families have been described in E. coli.32,33 reaction conditions were adjusted or different co-solvents were
In particular, ethanol-inducible ethanol dehydrogenase/ used while the reaction rates were comparable, all subsequent
acetaldehyde reductase, encoded by adhP or yddN gene was reactions were carried out in the presence of 2-butanol as
discovered relatively late due to inducibility of its activity by reactions with it resulted in highest product yield.
EtOH.34 Recently, AdhP crystal structure was reported.35 It was
found that presence of 0.017 M EtOH induced AdhP activity, Biotransformation of a range of a,a-substituted aromatic and
while the amount of EtOH in our reaction was 0.17 M.
heterocyclic nitroalkenes
The desired g-nitroaldehyde 1a was obtained in moderate
enantioselectivity (48% ee; Table 3; Entry I). It was previously
shown that specicity and control of stereochemical outcome of
biocatalytic reactions could be achieved by adjustment of reac-
tion conditions.36,37 For example, pentaerythritol tetranitrate
reductase enzyme scaffold was shown to be capable of generating
both enantiomeric products with improved enantiopurities by a
manipulation of the reaction conditions and/or in the presence
of one or two key mutations.36 Having in mind that the enzymatic
activity of 4-OT mostly relies on Pro1 residue29 and does not leave
too much room for mutational improvements, we attempted to
improve the stereoselectivity of Michael-type additions by
lowering the pH of the reaction buffer to 5.5 and by reducing the
reaction temperature to 20 C and 10 C. Overall, both types of
adjustments had a negative effect on 1a yield from the reaction
catalyzed by whole-cell Escherichia coli BL21 (4-OT), while no
improvement in ee values was observed (ESI Table 1†). This is in
contrast to results obtained with puried 4-OT when lowering the
pH from 7.0 to 5.5 in the addition reaction of acetaldehyde and b-
With the best conditions of reaction dened, we explored if
substrate scope could be expanded to substituted aromatic (2
and 3), heterocyclic (4 and 5) and nonaromatic (6) nitroalkenes
in the reaction with isobutanal (a) (Table 2).
p-Chloro-trans-b-nitrostyrene (2) and 3,4-(methylenedioxy)-b-
nitrostyrene (3), as well as (E)-2-(thiophen-2-yl)nitroethene (4)
and (E)-2-(furan-2-yl)nitroethene (5) were biotransformed into
corresponding nitroaldehydes, while the only substrate that
could not be utilized as acceptor in this reaction was (E)-2-
cyclohexyl-1-nitroethene (6) (Table 2; Entry VI). This was not
surprising due to signicant difference between benzene and
cyclohexane ring in their steric and electronic properties.
Hence, the aromatic nature of the ring was concluded necessary
for reaction to occur. While reaction with 1 was completed
within 4 h, longer reaction times (24 h) were required for het-
eroaromatic Michael adducts 4a and 5a (thiophen-2-yl and
furan-2-yl) to be formed (Table 2; Entry IV and V). Desired
product yields, as estimated from the GC analysis were
approximately 2-fold lower for 4a and 5a in comparison to 1a
ꢀ
ꢀ
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Table 2 Synthesis of g-nitroaldehydes through biotransformation of
different nitroalkenes (1–6) and isobutanal (a) by recombinant E. coli
BL21 (4-OT) using 5 g CDW Lꢂ1 at 28 ꢀC, pH 7.2 in 60 mL volume
Michael-type additions catalyzed by 4-OT whole-cell biocatalyst
(Fig. 1). Based on the results of the initial reaction with iso-
butanal, three different Michael acceptors 1, 4 and 5 were
included in this part of the study.
Entry Substratea
Product Reaction time (h) Yieldb (%)
When b-nitrostyrene was used as substrate, corresponding g-
nitroaldehydes 1b–d bearing an all-carbon quaternary center in
the a-position were obtained in good yields (Table 3; Entry II–
IV). It is worth mentioning that although depletion of the
conversions of substrates were 100%, isolated products yields
were usually lower in comparison to the yield estimated by GC
analysis, suggesting that percentage of the product was not
successfully recovered during the purication procedure.
Reactions with sterically demanding a,a-dialkyl aldehydes (b–d)
with b-nitrostyrene required longer reaction time and afforded
products with lower enantioselectivity (Table 3; Entry II–IV).
This was also the case when 4 and 5 were used as Michael
acceptors (Table 3; Entry VI and VIII–X). Unfortunately, corre-
sponding Michael adducts when (E)-2-(thiophen-2-yl)
nitroethene (4) was used as acceptor were not obtained in
reactions with b and c. The lower enantioselectivity in
comparison to the reaction when isobutanal (a) was used as
substrate may be due to the structural extension causing steric
effect and substrate conformation of aldehydes b–d or even
retro-Michael reaction followed by side processes. The trend
that substrate conformation governs enantioselectivity of the
process was previously observed in Michael additions of
unmodied aldehydes with nitroolens catalyzed by pyrrolidine
sulfonamide.15 Indeed, similar observation was reported by
Yoshida and co-authors when various a-branched and
unbranched aldehydes were tested as Michael donors in reac-
tion with b-nitrostyrene catalyzed by phenylalanine lithium salt,
when bulkier aldehydes required longer reaction times and
asymmetric a-branched aldehydes such as 2-phenyl-
propionaldehyde caused reduced enantioselectivity.23 It
appeared that L-phenylalanine lithium salt as catalyst afforded
g-nitroaldehydes in good yields with much higher enantiose-
lectivity in comparison to our biocatalytic approach using
whole-cells expressing 4-oxalocrotonate tautomerase, therefore
we have applied the reported organocatalytic method in the
synthesis of 1a–d, 4a, 4d and 5a–d for direct comparison (Table
3: Entries I0–X0).
I
1a
2a
4
71
18
II
48
III
3a
48
12
IV
V
4a
5a
24
24
37
39
VI
nrc
—
—
a
Nitroalkenes (1–6) were added to reaction as stock solution in 2-
b
c
butanol. Estimated yield based on GC-MS analysis. No reaction
occurred aer 72 h incubation.
with by-product of substrate reduction occurring at much
higher percentage between 30–40% (results not shown). On the
other side substituted aromatic acceptors 2 and 3 when utilized
in the reaction with isobutanal (a) even aer prolonged incu-
bation of 48 h afforded only 18 and 12% of desired g-nitro-
aldehydes products, respectively. The biotransformation
product mixture of these two substrates was very complex con-
taining high amounts untransformed starting material (40–
50%) and reduction and other degradation by-products (20–
30%). Overall, 2a and 3a were the least represented in the cor-
responding biotransformations mixtures, hampering their
successful isolation and purication, so 2 and 3 were not
included as substrates in the subsequent study. This was
surprising, as in the similar reaction using the same biocatalyst
and acetaldehyde as donor, p-chloro-trans-b-nitrostyrene (2) was
successfully transformed to 3-(4-chlorophenyl)-4-nitrobutanal
with 38% isolated product yield,30 suggesting the steric impor-
tance of the Michael donor in this reaction.
In direct comparison isolated product yields were generally
higher in synthesis using asymmetric organocatalytic approach
(Table 3; gray shaded rows) with ee values between 92 and 96%,
with reaction times comparable or longer and the reaction
medium was CH2Cl2. Interestingly, when 2-ethylbutanal (b) was
used as Michael donor, biocatalytic approach was superior in
terms of product yield and the reaction time (Table 3; Entry II
and II0). In the case when non-symmetrically branched aldehyde
d, slightly better diastereoselectivity of Michael adducts was
observed by NMR analysis in organocatalytic approach (Table 3;
Entry IV0, VI0 and X0). Great differences in two approaches,
especially in terms of enantioselectivity could be explained by
the fact that the lithium cation behaves as a Lewis acid to aid
the formation of enamine between the catalyst and aldehydes
Different branched aldehydes as donors in Michael-additions
catalyzed by 4-OT
It was explored if 2-ethylbutanal (b), cyclohexanecarbaldehyde and enables favourable transition state.23,38 However,
(c) and 2-methylpentanal (d) could be employed as donors in secondary amino acid L-proline and its lithium salt were not
a
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Table 3 Comparative synthesis of g-nitroaldehydes through transformation of a,b-substituted nitroalkenes and branched aldehydes by
recombinant tautomerase and using lithium salt of phenylalanine
Entry
Substratea
Product
Reaction time (h)
Isolated yield (%)
drb
eec (%)
I
1
1
1
1
1
1
1
1
4
4
4
4
5
5
5
5
5
5
5
5
1a
1a
1b
1b
1c
1c
1d
1d
4a
4a
4d
4d
5a
5a
5b
5b
5c
5c
5d
5d
4
24
16
72
20
24
20
24
24
24
24
24
24
24
36
72
36
24
36
24
60
52
70
31
57
41
59
58
34
78
32
70
37
60
10
12
35
69
27
67
—
—
—
—
—
—
64/36
74/26
—
48
94
9
I0
II
II0
92
17
95
III
III0
IV
IV0
V
d
—
—
10
94
—
—
2
96
8
94
6
V0
—
VI
VI0
VII
VII0
VIII
VIII0
IX
IX0
X
67/33
75/25
—
—
—
—
—
—
64/36
74/26
94
—
—
X0
a
Substrates were added to reaction as stock solution in 2-butanol. b Determined by 1H NMR. c Determined by chiral HPLC analysis using Chiralpak
d
IA. Not determined because of insufficient chromatographic separation of enantiomers.
able to catalyse this Michael-type addition, as reported in the nitroaldehydes have not been examined previously for antimi-
same study.23 As mentioned, catalytic mechanism of 4-OT in crobial activity and their cytotoxic properties have not been
Michael addition relies on promiscuous activity of this enzyme reported.
and terminal proline residue that participates in the formation
of a nucleophilic enamine intermediate.27
As g-nitroaldehydes were obtained in sufficient amounts
they were screened against a range of Gram-positive and Gram-
Other organocatalytic approaches were also employed in negative bacteria and exhibited no signicant antibacterial
synthesis of some of the g-nitroaldehydes obtained in this activity including Micrococcus luteus, Enterococcus faecalis,
study. 1-(2-Nitro-1-phenylethyl)cyclohexanecarbaldehyde (1c) Staphylococcus aureus, Klebsiella pneumoniae (results not
was previously obtained by organocatalysis.15,39–42 In most shown). However, antifungal activity against Candida albicans
cases product yields reported were between 31–51% which is was more prominent (Table 4).
comparable to our biocatalytic result but reaction times were
Due to structural similarity the activity of generated
about 3–5 times longer (70–96 h vs. 20 h). The best organo- aldehydes was compared to that of b-nitrostyrene as a known
catalytic approach to produce 1c with a reported yield of 90% antifungal agent.44 C. albicans MIC values of g-nitro-
and a reaction time of 24 h was performed using rather aldehydes were higher in comparison to the MIC value of 1,
specialised chiral amine/acid bifunctional catalyst (0.3 but they also exhibited much less cytotoxicity against human
equiv.).18
broblasts in vitro (Table 3). Nitrostyrene has also previously
This is the rst time that 2,2-diethyl-4-nitro-3-phenylbutanal been found to be cytotoxic on stomach cancer lines in doses
(1b), 2-methyl-2-(2-nitro-1-(thiophen-2-yl)ethyl)pentanal (4d), of 0.75 mg mLꢂ1 and to have adverse effects on the immune
2,2-diethyl-3-(furan-2-yl)-4-nitrobutanal (5b) and 2-(1-(furan-2- system.45 Of particular interest was 2,2-diethyl-4-nitro-3-
yl)-2-nitroethyl)-2-methylpentanal (5d) were synthesized phenylbutanal (1b) with good antifungal activity and cyto-
and characterized using biocatalytic and organocatalytic toxic effect exhibited in 2.5- and fold higher concentrations
approach.
than MIC values against fungal strain (Table 4). Still, MIC
values against C. albicans obtained for g-nitroaldehydes are
higher than those of antifungal agents clinically available
Antimicrobial properties and cytotoxicity of g-nitroaldehydes
such as nystatin which MIC against the same strain of C.
albicans was determined to be 5–10 mg mLꢂ1
.
However,
46
Pharmacological effects of GABA and analogs are usually
generated by interaction with both GABA(B) and gamma-
hydroxybutyric acid (GHB) receptors, as well as from the inu-
ence on other transmitter systems in the human brain.11 On the
other hand, b-nitrostyrene derivatives have shown potential
antibacterial activities.43 To the best of our knowledge, g-
nystatin and other clinically used antifungals also exhibit
potent cytotoxicity; therefore search for novel antifungal
agents is ongoing effort.47
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Table 4 In vitro antifungal and anti proliferative activity of nitrostyrene
and g-nitroaldehydes generated in this study against Candida albicans
and MRC5 fibroblast cell line
Bacterial media components, casamino acids, ampicillin,
isopropyl-b-D-thiogalactopyranoside (IPTG) were purchased
from Sigma-Aldrich (Munich, Germany) or Fisher Scientic
(Hampton, NH, USA) unless otherwise stated, and used without
further purication.
MICa (mg mLꢂ1
)
IC50b (mg mLꢂ1
)
Candida albicans
Compound
ATCC 10259
MRC5
Biocatalyst preparation and representative biocatalytic
reaction
1
10 ꢄ 1
100 ꢄ 3
40 ꢄ 1
150 ꢄ 4
60 ꢄ 5
170 ꢄ 7
40 ꢄ 2
50 ꢄ 4
100 ꢄ 5
80 ꢄ 3
200 ꢄ 6
0.5 ꢄ 0.1
50 ꢄ 1
100 ꢄ 2
60 ꢄ 1
50 ꢄ 2
60 ꢄ 2
60 ꢄ 2
40 ꢄ 1
40 ꢄ 2
80 ꢄ 2
30 ꢄ 1
1a
1b
1c
1d
4a
4d
5a
5b
5c
5d
Whole-cell biocatalyst (Escherichia coli BL21 (4-OT)) used in this
study was generated in our laboratory and recently described.30
Briey, this strain is recombinantly expressing 4-oxalocrotonate
tautomerase (4-OT) from Pseudomonas putida mt-2 with induc-
ible expression under control of T7 polymerase. For the
biotransformations, cultures were grown in M9 medium51
supplemented with glucose (1%, w/v), casamino acids (0.5%,
w/v) and ampicillin (50 mg mLꢂ1) at 30 ꢀC with shaking at
a
MIC values (minimal inhibitory concentrations) are calculated from 200 rpm. Once the culture had reached an optical density of 0.5
three independent experiments and are expressed as means ꢄ SD.
(600 nm; Spectophotometer Ultrospec 3300pro, Amersham
Biosciences), cells were induced with 0.1 mM IPTG at 28 ꢀC.
Aer 12 h incubation, wet cell pellets were resuspended to a
concentration of cell dry weight (CDW) of 5 g Lꢂ1 (OD600 ¼ 20) in
20 mM phosphate buffer pH 7.2. Cell free extracts of the E. coli
BL21 (4-OT) cells were prepared in a similar way, by disrupting
cell membrane using Ultrasonicator (5 cycles of 10 s pulse and
20 s pause) and removing cell debris using centrifugation.
The whole cell biotransformation was generally carried out
in 250 mL Duran Schott glass bottles with screw type plastic lids
containing 60 mL cells suspension at 28 ꢀC with shaking at 150
rpm. Unless otherwise stated, a,b-unsaturated nitroalkenes (1–
6) were added sequentially as described previously30 to a nal
concentration of 2 mM (18 mg, 0.12 mmol) from a 200 mM
stock solution in 2-butanol and aldehydes a–d (1.2 mmol) were
added to 20 mM nal concentration. Samples (800 mL) were
withdrawn from the reaction over time, centrifuged at 13 000 ꢃ
g for 5 min and supernatants analyzed spectrophotometrically
as previously described, following the depletion of 1 by reduc-
tion of absorbance at 320 nm (3 ¼ 14.4 mMꢂ1 cmꢂ1).29
b
IC50 values (concentrations at which 50% cell growth inhibition
occurs) are calculated from three independent experiments and are
expressed as means ꢄ SD.
Conclusion
Previously reactions of 4-oxalocrotonate tautomerase whole cell
biocatalyst with acetaldehyde and a range of b-nitrostyrenes
were rapid and yielded products with high enantiopurity.30
During this study the scope of the biocatalytic reaction based on
4-OT was signicantly widened, so that biotransformations with
both acyclic and cyclic branched aldehydes afforded good route
to g-nitroaldehydes containing quaternary carbon in a-position
but showed reduced enantiopurity. Although biocatalytic route
offers mild reaction conditions and thereby safer and environ-
mentally friendlier synthesis, we have also shown that asym-
metric organocatalytic approach in synthesis of these products
is superior in terms of enantiopurity. Regardless of the applied
approach, g-nitroaldehydes with an all carbon quaternary
center in a-position provide an easy production route to novel g-
amino acids, g-butyrolactones and substituted pyrrolidines. In
addition to their structural similarity to GABA precursors, newly
synthesised g-nitroaldehydes were shown to have other bio-
logical activities such as antifungal.
To extract and purify the products from the biotransforma-
tion, the reaction mixture was extracted with ethylacetate (2 ꢃ
100 mL). The combined organic extract was washed with brine
(100 mL), dried over anhydrous MgSO4. Aer ltration and
removal of the solvent under reduced pressure, the residue was
puried by dry ash column chromatography (silica gel),
eluting with petroleum ether–ethylacetate mixture in gradient
(from 7 : 3 to 9 : 1) to afford pure nitroaldehydes.
General procedure for the synthesis of g-nitroaldehydes
from the corresponding aldehydes catalysed by L-phenylalanine
lithium salt was carried out using reported procedures.22,23
Experimental
Materials
b-Nitrostyrene (1), 3,4-(methylenedioxy)-b-nitrostyrene (3), (E)-2-
(thiophen-2-yl)nitroethene (4), isobutanal, 2-ethylbutanal,
cyclohexane-carbaldehyde and 2-methylpentanal were obtained
Analytical methods (HPLC)
from Sigma Aldrich (Munich, Germany). Nitroalkenes 2,48 549 The enantiomeric excess was determined by HPLC (Agilent
and 650 were prepared according to the published procedures. Technologies, HP110) with Chiralpak IA column (Chiral Tech-
Ethylacetate, ethanol and other solvents were of HPLC reagent nologies Europe, Cedex, France) at 210 nm for all samples.
grade and purchased from Fisher Scientic (Hampton, NH, Racemic mixture of the samples was prepared chemically from
USA). All solvents were freshly distilled before use.
the corresponding aldehydes using reported procedure.48
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NMR spectra were recorded on a Varian Gemini 200 (1H 0.92 (minor isomer) and 0.85 (major isomer) (t, J ¼ 7.2 and 7.1
NMR at 200 MHz, 13C NMR at 50 MHz, for samples in deuter- Hz, 3H). 13C NMR (125 MHz, CDCl3) d 204.6 (major isomer) and
ated chloroform), and on Bruker Avance III 500 (1H NMR at 500 204.2 (minor isomer), 137.8, 127.93 (major isomer) and 127.86
MHz, 13C NMR at 125 MHz). Chemical shis are expressed in (minor isomer), 126.9, 125.58 (minor isomer) and 125.46 (major
ppm (d) using tetramethylsilane as internal standard, coupling isomer), 78.2, 51.8 (major isomer) and 51.1 (minor isomer), 44.4
constants (J) are in Hz. IR spectra were recorded on a Nicolet (minor isomer) and 42.4 (major isomer), 37.6 (major isomer)
6700 FT instrument, and are expressed in cmꢂ1. Mass spectra and 36.4 (minor isomer), 17.1 (minor isomer) and 17.0 (major
were obtained on Agilent technologies 6210 TOF LC/MS isomer), 16.0, 14.5 (minor isomer) and 14.3 (major isomer). IR
instrument (LC: series 1200). GC/MS analyses were performed nmax: 2963, 2935, 1723, 1556. HRMS (ESI): m/z calcd for
on Agilent technologies 7890A-5975C inert XL EI CI instrument. C12H21N2O3S: 273.1267 [M + NH4]+; found 273.1270.
3-(Furan-2-yl)-2,2-dimethyl-4-nitrobutanal (5a)23 1H NMR
(200 MHz, CDCl3) d 9.53 (s, 1H), 7.38 (d, J ¼ 1.6 Hz, 1H), 6.32
Characterization of g-nitroaldehydes
(dd, J1 ¼ 1.6 Hz, J2 ¼ 3.2 Hz, 1H), 6.20 (d, J ¼ 3.2 Hz, 1H), 4.76
2,2-Dimethyl-4-nitrophenylbutanal (1a)23 1H NMR (200 MHz, (dd, J1 ¼ 10.9 Hz, J2 ¼ 12.9 Hz, 1H), 4.59 (dd, J1 ¼ 4.1 Hz, J2 ¼
CDCl3) d 9.53 (s, 1H), 7.40–7.13 (m, 5H), 4.92–4.64 (m, 2H), 3.78 12.9 Hz, 1H), 3.92 (dd, J1 ¼ 4.1 Hz, J2 ¼ 10.9 Hz, 1H), 1.18 (s, 3H),
(dd, J1 ¼ 11 Hz, J2 ¼ 4.4 Hz, 1H), 1.14 (s, 3H), 1.01 (s, 3H); 13C 1.05 (s, 3H); HPLC (Chiralpak IA, i-propanol–heptane ¼ 10/90,
NMR (50 MHz, CDCl3) d 204.3, 135.3, 129.0, 128.7, 128.1, 76.3, ow rate 1 mL minꢂ1, l ¼ 210 nm): tmajor ¼ 6.63 min, tminor
48.3, 48.2, 21.6, 18.8; HPLC (Chiralpak IA, i-propanol–heptane 7.20 min.
¼
¼ 20/80, ow rate 1 mL minꢂ1, l ¼ 210 nm): tmajor ¼ 2.98 min,
2,2-Diethyl-3-(furan-2-yl)-4-nitrobutanal (5b) 1H NMR (200
MHz, CDCl3) d 9.61 (s, 1H), 7.36 (dd, J1 ¼ 0.6 Hz, J2 ¼ 1.8 Hz 1H),
tminor ¼ 4.01 min; IR nmax: 2975, 2935, 1725, 1554.
1
2,2-Diethyl-4-nitro-3-phenylbutanal (1b) H NMR (200 MHz, 6.31 (dd, J1 ¼ 1.8 Hz, J2 ¼ 3.2 Hz, 1H), 6.21 (dd, J1 ¼ 0.6 Hz, J2 ¼
CDCl3) d 9.58 (s, 1H), 7.38–7.11 (m, 5H), 4.85–4.58 (m, 2H), 3.33 3.2 Hz, 1H), 4.81–4.62 (m, 2H), 3.88 (dd, J1 ¼ 4.9 Hz, J2 ¼ 10.0
(t, J ¼ 7.4 Hz, 1H), 1.80–0.86 (m, 10H). 13C NMR (50 MHz, CDCl3) Hz, 1H), 1.72–1.51 (m, 4H), 0.91 (t, J ¼ 7.5 Hz, 3H), 0.89 (t, J ¼ 7.5
d 207.8, 135.2, 129.1, 128.9, 128.8, 128.5, 128.1, 127.4, 48.1, 33.4, Hz, 3H). 13C NMR (50 MHz, CDCl3). d 206.0, 149.4, 142.4, 110.4,
23.5, 22.6, 7.9, 7.5. IR nmax: 2972, 2941, 1718, 1553. HRMS (ESI): 109.8, 75.2, 53.7, 41.0, 23.6, 22.9, 7.9, 7.6. IR nmax: 2971, 2936,
m/z calcd for C14H23N2O3: 267.1703 [M + NH4]+; found 267.1693. 2883, 1719, 1555. HRMS (ESI): m/z calcd for C12H21N2O4:
HPLC (Chiralpak IA, i-propanol–heptane ¼ 30/70, ow rate 1 257.1496 [M + NH4]+; found 257.1501. HPLC (Chiralpak IA, i-
mL minꢂ1, l ¼ 210 nm): tmajor ¼ 3.87 min, tminor ¼ 4.72 min.
propanol–heptane ¼ 10/90, ow rate 1 mL minꢂ1, l ¼ 210 nm):
1-(2-Nitro-1-phenylethyl)cyclohexanecarbaldehyde (1c)18 1H tminor ¼ 6.37 min, tmajor ¼ 6.92 min.
NMR (200 MHz, CDCl3) d 9.56 (s, 1H), 7.63–7.02 (m, 5H), 4.90–
1-(1-(Furan-2-yl)-2-nitroethyl)cyclohexanecarbaldehyde (5c)42
4.58 (m, 2H), 3.54 (dd, J1 ¼ 10 Hz, J2 ¼ 5.6 Hz, 1H), 2.39–0.85 (m, 1H NMR (200 MHz, CDCl3) d 9.59 (s, 1H), 7.37 (d, J ¼ 1.8 Hz, 1H),
10H), 13C NMR (50 MHz, CDCl3). d 207.3, 134.8, 129.1, 128.7, 6.31 (dd, J1 ¼ 1.8 Hz, J2 ¼ 3.2 Hz, 1H), 6.20 (d, J ¼ 3.2 Hz, 1H),
128.1, 76.3, 51.2, 50.4, 30.9, 29.6, 25.0, 22.6, 22.5; IR nmax: 2933, 4.76–4.56 (m, 2H), 3.68 (dd, J1 ¼ 4.8 Hz, J2 ¼ 10.2 Hz, 1H), 2.11–
2856, 1719, 1553. HPLC (Chiralpak IA, i-propanol–heptane ¼ 1.16 (m, 10H). HPLC (Chiralpak IA, i-propanol–heptane ¼ 10/
30/70, ow rate 1 mL minꢂ1, l ¼ 210 nm): tmajor ¼ 4.13 min, 90, ow rate 1 mL minꢂ1, l ¼ 210 nm): tmajor ¼ 5.79 min,
tminor ¼ 4.89 min.
tminor ¼ 6.09 min.
2-Methyl-2-(2-nitro-1-phenylethyl)pentanal (1d)18 1H NMR
2-(1-(Furan-2-yl)-2-nitroethyl)-2-methylpentanal (5d) 1H NMR
(500 MHz, CDCl3) d 9.54 (major isomer) and 9.52 (minor isomer) (500 MHz, CDCl3) d 9.55 (minor isomer) and 9.49 (major isomer)
(s, 1H), 7.34–7.27 (m, 3H), 7.21–7.16 (m, 2H), 4.85 (minor (s, 1H), 7.38 (major isomer) and 7.35 (minor isomer) (dd, J ¼ 0.6,
isomer) and 4.84 (major isomer) (dd, J ¼ 11.3, 13.1 and 11.5, 1.8 and 0.6, 1.9 Hz, 1H), 6.32 (major isomer) and 6.30 (minor
13.0 Hz, 1H), 4.76 (minor isomer) and 4.63 (major isomer) (dd, J isomer) (dd, J ¼ 1.8, 3.2 and 1.9, 3.2 Hz, 1H), 6.22 (major isomer)
¼ 4.3, 13.1 and 3.9, 13.0 Hz, 1H), 3.79 (major isomer) and 3.77 and 6.20 (minor isomer) (dd, J ¼ 0.6, 3.2 and 0.6, 3.2 Hz, 1H),
(minor isomer) (dd, J ¼ 3.9, 11.4 and 4.3, 11.2 Hz, 1H), 1.63–1.17 4.76 (minor isomer) and 4.73 (major isomer) (dd, J ¼ 11.1, 13.0
(m, 4H), 1.11 (major isomer) and 1.10 (minor isomer) (s, 3H), 0.9 and 11.3, 12.8 Hz, 1H), 4.64 (minor isomer) and 4.55 (major
(minor isomer) and 0.84 (major isomer) (t, J ¼ 7.2 and 6.9 Hz, isomer) (dd, J ¼ 3.8, 13.0 and 3.6, 12.8 Hz, 1H), 3.99 (major
3H).
isomer) and 3.90 (minor isomer) (dd, J ¼ 3.6, 11.3 and 3.8, 11.1
2,2-Dimethyl-4-nitro-3-(thiophen-2-yl)butanal (4a)23 1H NMR Hz, 1H), 1.59–1.18 (m, 4H), 1.17 (major isomer) and 1.11 (minor
(200 MHz, CDCl3) d 9.54 (s, 1H), 7.27–7.22 (m, 1H), 7.00–6.91 isomer) (s, 3H), 0.91 (minor isomer) and 0.85 (major isomer) (t, J
(m, 2H), 4.75–4.59 (m, 2H), 4.14 (dd, J1 ¼ 4.9 Hz, J2 ¼ 10.0 Hz, ¼ 7.2 and 7.1, 3H) 13C NMR 204.1, 149.7, 142.7, 110.6, 109.2,
1H), 1.21 (s, 3H), 1.09 (s, 3H); HPLC (Chiralpak IA, i-propanol– 75.3 (major isomer) and 74.7 (minor isomer), 51.5 (major
heptane ¼ 10/90, ow rate 1 mL minꢂ1, l ¼ 210 nm): tminor
8.08 min, tmajor ¼ 7.82 min.
¼
isomer) and 51.1 (minor isomer), 42.7 (minor isomer) and 40.5
(major isomer), 37.6 (major isomer) and 36.6 (minor isomer),
2-Methyl-2-(2-nitro-1-(thiophen-2-yl)ethyl)pentanal (4d) 1H 17.1 (minor isomer) and 16.9 (major isomer), 16.5, 14.6 (minor
NMR (500 MHz, CDCl3) d 9.54 (s, 1H), 7.26–7.22 (m, 1H), 6.98– isomer) and 14.4 (major isomer). IR nmax: 2964, 2935, 2874,
6.89 (m, 2H), 4.78–4.60 (m, 2H), 4.20 (major isomer) and 4.12 1723, 1557. HRMS (ESI): m/z calcd for C12H21N2O4: 257.1496 [M
(minor isomer) (dd, J ¼ 3.8, 11.1 and 4.5, 10.4 Hz, 1H), 1.69–1.22 + NH4]+; found 257.1500.
(m, 4H), 1.20 (major isomer) and 1.17 (minor isomer) (s, 3H),
60508 | RSC Adv., 2014, 4, 60502–60510
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RSC Advances
14 J. Xiang, E.-X. Sun, C.-X. Lian, W.-C. Yuan, J. Zhu, Q. Wang
and J. Deng, Tetrahedron, 2012, 68, 4609–4620.
15 J. Wang, H. Li, B. Lou, L. Zu, H. Guo and W. Wang, Chem.–
Eur. J., 2006, 12, 4321–4332.
16 N. Mase, in Science of Synthesis Asymmetric Organocatalysis,
ed. B. List and K. Maruoka, Thieme, Stuttgart, 2012, pp.
135–216.
17 L. Zhao, J. Shen, D. Liu, Y. Liu and W. Zhang, Org. Biomol.
Chem., 2012, 10, 2840–2846.
18 N. Mase, R. Thayumanavan, F. Tanaka and C. F. r. Barbas,
Org. Lett., 2004, 6, 2527–2530.
19 N. S. Chowdari, J. T. Suri and C. F. Barbas, Org. Lett., 2004, 6,
2507–2510.
20 M. P. Lalonde, Y. Chen and E. N. Jacobsen, Angew. Chem., Int.
Ed., 2006, 45, 6366–6370.
21 D. Almasi, D. A. Alonso and C. Najera, Tetrahedron:
Asymmetry, 2007, 18, 299–365.
22 A. Sato, M. Yoshida and S. Hara, Chem. Commun., 2008,
6242–6244.
In vitro antimicrobial and cytotoxic assays
To test antimicrobial properties of synthesized g-nitro-
aldehydes a range of bacterial strains from the American Type
Culture Collection (ATCC) including Micrococcus luteus ATCC
379, Enterococcus faecalis ATCC 29212, Staphylococcus aureus
ATCC 25923, Klebsiella pneumoniae ATCC 13883, Pseudomonas
aeruginosa ATCC 27853 and Candida albicans ATCC 10259 in
standard Kirby-Bauer disc diffusion assay. Minimal inhibitory
concentrations (MIC) against C. albicans ATCC 10259 were
determined in liquid culture in 96-well microtiter plate assay.52
Cytotoxicity of the compounds was assessed against human
lung broblast MRC5 cell line obtained from ATCC using MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
reduction assay.53 The MTT assay was performed aer 24 h
treatment with compounds three times in three replicates and
the IC50 values (concentrations at which 50% cell growth inhi-
bition occurred) were calculated in comparison to control
(untreated cells) that were arbitrarily set to 100%.
23 M. Yoshida, A. Sato and S. Hara, Org. Biomol. Chem., 2010, 8,
3031–3036.
24 R. Kastl and H. Wennemers, Angew. Chem., Int. Ed., 2013, 52,
7228–7232.
25 B. Ni, Q. Zhang and A. D. Headley, Green Chem., 2007, 9, 737–
739.
26 R. C. Wende and P. R. Schreiner, Green Chem., 2012, 14,
1821–1849.
27 E. M. Geertsema, Y. Miao, P. G. Tepper, P. de Haan,
E. Zandvoort and G. J. Poelarends, Chem.–Eur. J., 2013, 19,
14407–14410.
28 Y. Miao, E. M. Geertsema, P. G. Tepper, E. Zandvoort and
G. J. Poelarends, ChemBioChem, 2013, 14, 191–194.
29 E. Zandvoort, E. M. Geertsema, B. J. Baas, W. J. Quax and
G. J. Poelarends, Angew. Chem., Int. Ed., 2012, 51, 1240–1243.
Acknowledgements
This work was supported by Ministry of Education and Science,
Republic of Serbia grants 173048 and 172002. We thank Chiral
Technologies Europe and Mrs Christina Suteu for generous gi
of analytical HPLC column CHIRALPACK IA 250 and Dr Branka
Ivkovic (University of Belgrade, Faculty of Pharmacy) for help
with HPLC analysis. We also acknowledge Chemistry and
Molecular Sciences and Technologies COST Action CM1303. We
ˇ
ˇ
´
dedicate this work to Professor Zivorad Cekovic on the occasion
his 80th birthday.
Notes and references
1 J. P. Das and I. Marek, Chem. Commun., 2011, 47, 4593–4623. 30 T. Narancic, J. Radivojevic, P. Jovanovic, D. Francuski,
2 U. Scheffler and R. Mahrwald, Chem.–Eur. J., 2013, 19, 14346–
14396.
3 M. Bella and T. Gasperi, Synthesis, 2009, 10, 1583–1614.
M. Bigovic, V. Maslak, V. Savic, B. Vasiljevic,
K. E. O'Connor and J. Nikodinovic-Runic, Bioresour.
Technol., 2013, 142, 462–468.
4 B. Wang and Y. Q. Tu, Acc. Chem. Res., 2011, 44, 1207–1222. 31 P. Jovanovic, S. Jeremic, L. Djokic, V. Savic, J. Radivojevic,
5 K. Patora-Komisarska, M. Benohoud, H. Ishikawa,
D. Seebach and Y. Hayashi, Helv. Chim. Acta, 2011, 94, 719–
745.
6 P. Garcia-Garcia, A. Ladepeche, R. Halder and B. List, Angew.
Chem., Int. Ed., 2008, 47, 4719–4721.
7 G. Choudhary and R. K. Peddinti, Green Chem., 2011, 13,
276–282.
8 S. Mukherjee, J. W. Yang, S. Hoffmann and B. List, Chem.
Rev., 2007, 107, 5471–5569.
V. Maslak, B. Ivkovic, B. Vasiljevic and J. Nikodinovic-
Runic, Enzyme Microb. Technol., 2014, 60C, 16–23.
32 R. Ladenstein, J. O. Winberg and J. Benach, Cell. Mol. Life
Sci., 2008, 65, 3918–3935.
33 A. Pick, B. Ruhmann, J. Schmid and V. Sieber, Appl.
Microbiol. Biotechnol., 2013, 97, 5815–5824.
34 J. Shafqat, J.-O. Hoog, L. Hjelmqvist, U. C. T. Oppermann,
C. Ibanez and H. Jornvall, Eur. J. Biochem., 1999, 263, 305–
311.
9 I. Zenz and H. Mayr, J. Org. Chem., 2011, 76, 9370–9378.
10 M. N. Aboul-Enein, A. A. El-Azzouny, O. A. Saleh and
Y. A. Maklad, Mini-Rev. Med. Chem., 2012, 12, 671–700.
35 L. M. Thomas, A. R. Harper, W. A. Miner, H. O. Ajufo,
K. M. Branscum, L. Kao and P. A. Sims, Acta Crystallogr.,
Sect. F: Struct. Biol. Cryst. Commun., 2013, 69, 730–732.
11 H. Andresen, B. E. Aydin, A. Mueller and S. Iwersen- 36 A. Fryszkowska, H. Toogood, M. Sakuma, G. M. Stephens,
Bergmann, Drug Test. Anal., 2011, 3, 560–568.
12 K. Gajcy, S. Lochynski and T. Librowski, Curr. Med. Chem.,
2010, 17, 2338–2347.
13 N. Ono, The nitro group in organic synthesis, Wiley-VCH, New
York, NY, 2001.
J. M. Gardiner and N. S. Scrutton, Catal.: Sci. Technol.,
2011, 1, 948–957.
37 A. Fryszkowska, H. S. Toogood, D. Mansell, G. Stephens,
J. M. Gardiner and N. S. Scrutton, FEBS J., 2012, 279, 4160–
4171.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 60502–60510 | 60509

View Article Online
RSC Advances
Paper
38 M. Yoshida, N. Kitamikado, H. Ikehara and S. Hara, J. Org. 46 N. Stankovic, L. Senerovic, Z. Bojic-Trbojevic, I. Vuckovic,
Chem., 2011, 76, 2305–2309.
39 J.-F. Bai, X.-Y. Xu, Q.-C. Huang, L. Peng and L.-X. Wang,
Tetrahedron Lett., 2010, 51, 2803–2805.
40 T. C. Nugent, A. Bibi, A. Sadiq, M. Shoaib, M. N. Umar and
F. N. Tehrani, Org. Biomol. Chem., 2012, 10, 9287–9294.
41 T. C. Nugent, M. Shoaib and A. Shoaib, Org. Biomol. Chem.,
2011, 9, 52–56.
42 L. Tuchman-Shukron, S. J. Miller and M. Portnoy, Chem.–
Eur. J., 2012, 18, 2290–2296.
L. Vicovac, B. Vasiljevic and J. Nikodinovic-Runic, J. Appl.
Microbiol., 2013, 1297–1306.
47 L. Ostrosky-Zeichner, A. Casadevall, J. N. Galgiani, F. C. Odds
and J. H. Rex, Nat. Rev. Drug Discovery, 2010, 9, 719–727.
48 O. Andrey, A. Alexakis and G. Bernardinelli, Org. Lett., 2003,
5, 2559–2561.
49 S. Y. Mahmood, M.-C. Lallemand, L. Sader-Bakaouni,
O. Charton, P. Verite, H. Dufat and F. Tillequin,
Tetrahedron, 2004, 60, 5105–5110.
43 N. Milhazes, R. Calheiros, M. P. M. Marques, J. Garrido, 50 A. Duursma, A. J. Minnaard and B. L. Feringa, Tetrahedron,
M. N. D. S. Cordeiro, C. Rodrigues, S. Quinteira, C. Novais, 2002, 58, 5773–5778.
L. Peixe and F. Borges, Bioorg. Med. Chem., 2006, 14, 4078– 51 J. Sambrook and W. D. Russell, Molecular cloning a laboratory
4088.
manual, Cold Spring Harbour Laboratory Press, Cold Spring
44 E. E. Evans, R. F. Haines, A. C. Curtis, F. C. Bocobo,
Harbour, New York, USA, 2001.
W. D. Block and E. R. Harrell, J. Invest. Dermatol., 1956, 27, 52 J. T. Casey, C. O'Cleirigh, P. Walsh and D. G. O'Shea, J.
43–48. Microbiol. Methods, 2004, 58, 327–334.
45 K. C. Carter, Y. S. Finnon, N. N. Daeid, D. C. Robson and 53 M. B. Hansen, S. E. Nielsen and K. Berg, J. Immunol. Methods,
R. Waddell, Immunopharmacol. Immunotoxicol., 2002, 24,
187–197.
1989, 119, 203–210.
60510 | RSC Adv., 2014, 4, 60502–60510
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