A synergic blend of newly isolated pseudomonas mandelii KJLPB5 and [hmim]Br for chemoselective 2° aryl alcohol oxidation in H2O2: Synthesis of aryl ketone or aldehydes via sequential dehydration-oxidative C=C cleavage

Article abstract of DOI:10.1007/s10562-010-0542-6

Pseudomonas mandelii KJLPB5 is reported for the oxidation of aryl alcohols in ionic liquid [hmim]Br (1-hexyl-3-methyl imidazolium bromide) with H ₂O₂. With a slight alteration of reaction conditions, the developed protocol leads either to (i) chemoselective oxidation of 2° aryl alcohols over 1° and aliphatic counterparts or (ii) direct one pot-two step sequential conversion of 2° aryl alcohols into corresponding one or two carbons shorter aryl aldehydes through oxidative cleavage pathway, thus providing a new facet to metal-free oxidations. The key operational parameters such as substrate concentration, incubation temperature, incubation time, ionic liquid type and ionic liquid concentration are also optimized. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10562-010-0542-6

Catal Lett (2011) 141:616–622
DOI 10.1007/s10562-010-0542-6
A Synergic Blend of Newly Isolated Pseudomonas mandelii
KJLPB5 and [hmim]Br for Chemoselective 2° Aryl Alcohol
Oxidation in H O : Synthesis of Aryl Ketone or Aldehydes
2
2
via Sequential Dehydration-Oxidative C=C Cleavage
Nandini Sharma
Richa Salwan Ramesh C. Kasana
Arun K. Sinha
•
Upendra K. Sharma
•
•
•
Received: 9 September 2010 / Accepted: 19 December 2010 / Published online: 5 January 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Pseudomonas mandelii KJLPB5 is reported for
the oxidation of aryl alcohols in ionic liquid [hmim]Br
1 Introduction
(
1-hexyl-3-methyl imidazolium bromide) with H O . With
2
The carbonyl group is one of the most prevalent functional
groups and is entailed in many important chemical reac-
tions. Generally, alkenes and alcohols are used as precur-
sors for the generation of carbonyls [1–5], and selective
oxidation of alcohols to carbonyl containing moieties
remains one of the fundamental processes in organic syn-
thesis [6–8]. A plethora of reagents are available for this
interconversion, but most of these are often expensive,
toxic and must be used in stoichiometric quantities [9, 10].
Consequently, both from the environmental and economi-
cal points of view, catalytic oxidations particular involving
clean oxidants such as O and H O are promising [11–18].
2
a slight alteration of reaction conditions, the developed
protocol leads either to (i) chemoselective oxidation of 2°
aryl alcohols over 1° and aliphatic counterparts or (ii)
direct one pot-two step sequential conversion of 2° aryl
alcohols into corresponding one or two carbons shorter aryl
aldehydes through oxidative cleavage pathway, thus pro-
viding a new facet to metal-free oxidations. The key
operational parameters such as substrate concentration,
incubation temperature, incubation time, ionic liquid type
and ionic liquid concentration are also optimized.
2
2 2
Keywords Biocatalysis ꢀ Pseudomonas mandelii ꢀ
Ionic liquid ꢀ Alkene cleavage ꢀ Oxidation
On the similar grounds, catalysis in ionic liquids (IL) as an
alternative to organic solvents is gaining significance due
to their negligible vapour-pressure, excellent solvent
properties and high stability [19, 20].
Furthermore, in the heightened context of developing
green chemical methodologies, dynamic efforts to exploit
the potential of biocatalysts (whole cell as well as
enzymes) is on rise due to their simple processing
requirements, high selectivity and mild reaction conditions.
A number of biocatalytic reports are available for the
synthesis of carbonyls either by oxidation of alcohols
IHBT Communication No. 2060.
Electronic supplementary material The online version of this
article (doi:10.1007/s10562-010-0542-6) contains supplementary
material, which is available to authorized users.
[
21–24] or by oxidative alkene cleavage [25–27]. Still, the
development of chemoselective methods that efficiently
discriminate among various functional groups remains
challenging. Our group has been involved in the explora-
tion of practical green chemistry procedures for various
organic transformations [21, 28–30]. In continuation of our
interest in the use of ionic liquids in biocatalytic synthesis
and taking into account the encouraging results of our
previous study on alcohol oxidation with lipase and
[hmim]Br [21], we were interested to explore microbial
N. Sharma ꢀ U. K. Sharma ꢀ A. K. Sinha (&)
Natural Plant Products Division, Institute of Himalayan
Bioresource Technology (CSIR), Palampur,
Himachal Pradesh 176061, India
e-mail: aksinha08@rediffmail.com
R. Salwan ꢀ R. C. Kasana
Hill Area Tea Science Division, Institute of Himalayan
Bioresource Technology (CSIR), Palampur,
Himachal Pradesh 176061, India
1
23

Pseudomonas mandelii KJLPB5 Catalyzed Oxidations in Ionic Liquid
617
catalyst from Western Himalayan region for metal free
oxidative transformations, especially those involving user
withdrawn periodically and extracted with ethyl acetate.
Organic extracts were filtered, dried over anhydrous
sodium sulphate and concentrated at 40 °C in vacuo to
remove solvent. 1 mL of HPLC grade methanol (Merck)
was added to the concentrated extract and filtered through
0.22 lM filter prior to analysis.
friendly reagent H O2 which generally requires metal
2
activation [21]. Such whole cell transformations are pref-
erable as they eliminate the need for enzyme purification
and coenzyme addition/regeneration [31].
Thus herein, we report a new bacterial strain Pseudo-
monas mandelii KJLPB5 for oxidation of aryl alcohols. The
developed protocol uses culture supernate of P. mandelii
KJLPB5 in IL [hmim]Br with 30% H O as oxidant. Besides,
For the preparation of culture supernate, culture having
OD550 0.8 were harvested, centrifuged at 10,0009g at 4 °C
for 25 min and supernatant (culture supernate) was sepa-
rated from the pellet. The pellet constituting resting cells
was re-suspended in 50 mM tris HCl buffer (pH 7) and
stored at 4 °C till further experiments. The protein content
of culture supernate was determined according to Bradford
method using BSA as standard.
2
2
the same protocol provides a novel and facile approach for
direct conversion of 2° aryl alcohols into corresponding one
or two carbon shorter aryl aldehydes (Scheme 1), which is
the first report of such bioconversions.
2.3 Analysis of Biotransformed Products
2
Experimental
Analysis was performed using a Shimadzu HPLC (Model
LC-20AT pump, DGU-20A5 degasser) equipped with auto
sampler (SIL-20AC), photo diode array detector (CBM-20A;
Shimadzu, Kyoto, Japan) and interfaced with IBM Pentium 4
personal computer. The separation was performed on a
Purospher star RP-18e column (150 9 4.6 mm id, 5 lM;
Merck) at 30 °C. The mobile phase consisted of (A) 0.05%
2
.1 Materials
Substrates were obtained from commercial sources (Merck
or Acros). Ionic liquid ([hmim]Br) was synthesized
according to reported method [31–33]. The solvents used
for isolation/purification of compounds were obtained from
Merck and used without further purification.
TFA (Trifluoroacetic acid) in H O and (B) methanol/aceto-
2
nitrile (in 70/30 v:v) with gradient elution (0–5 min, 40–70%
B; 5–10 min, 70–100% B; 10–12 min, 100–40% B;
2
.2 General Procedure
-1
12–20 min, 40% B) with a flow rate of 1 mL min . Analysis
Experiments were carried out in duplicate. Bacterial cul-
tures used in the study were isolated from the soils of
Western Himalayan region by following standard proce-
dures [34]. The strains capable of oxidation were rapidly
screened through HPLC analysis. Isolates found positive
for bioconversion of 4-methoxyphenylpropanol (1a) in
initial screening were grown in nutrient broth (NB) med-
ium at 28 °C and 220 rpm. 1a was added in final concen-
wavelength was set at 280 nm. The quantification was per-
formed using external standard method. GC–MS analysis was
1
undertaken using Shimadzu-2010. H (300 MHz) and
13
C
(75.4 MHz) NMR spectra were recorded on Bruker Avance-
300 spectrometer wherever mentioned.
2.4 Identification of Bacterial Strain
-
1
tration of 1–3 g L
and incubated at 20–40 °C and
Strain KJLPB5 showing oxidation and oxidative alkene
cleavage activity was then subsequently identified based on
2
20 rpm for 24, 36, 48, 72 and 96 h. Samples were
Scheme 1 2° aryl alcohol
oxidations catalyzed by cell free
supernate of P. mandelii
O
P. mandelii strain KJLPB5
R'
KJLPB5 in [hmim]Br with
30% H O , [hmim]Br
2 2
2
H O
2
R
(yield upto 46%)
OH
R'
R
R= OCH , Cl, NO , Br
3
2
R'= CH , CH CH etc.
3
2
3
CHO
1
2
. [hmim]Br, MW
. P. mandelii strain KJLPB5
R
3
0% H O
(yield upto 85%)
2
2
123

6
18
N. Sharma et al.
its 16S rDNA sequencing [35]. The primers used for
0
exogenous nature of the enzyme. So this culture supernate
was inevitably used for further reaction. The protein con-
-1
0
amplification were 5 -AGAGTTTGATCATGGCTC-3 and
0
0
5
-GTTACCTTGTTACGACTT-3 . Amplification reaction
was performed in a 50 lL reaction volume containing 1 lL
5 U) of Taq DNA polymerase (Sigma), 5 lL of 109 Taq
tent of culture supernate was found to be 0.064 mg mL
(For SDS page analysis, see supplementary data).
(
buffer, 1 lL of 10 mM dNTPs mix, 1 lL of each 10 lM
primer and 50–100 ng of template DNA. The reaction was
performed in a thermal cycler for 4 min at 94 °C and then
subjected to 35 amplification cycles of 20 s at 94 °C, 1 min
at 52 °C, for 2 min at 72 °C followed by a final extension
step of 8 min at 72 °C. The amplified PCR product was
electrophoresed on 0.8% agarose gel and amplicon of
3.3 Optimization of Biotransformation Procedure
Optimization experiments for the conversion of 1a into
4-methoxyphenylpropanone (1b) were carried out to find the
best suitable conditions. [hmim]Br was initially taken as
ionic liquid of choice based on the encouraging results of our
previous study [21]. To begin with, reaction mixture
(0.12 mM substrate ? 0.5 mL IL ? 6 equiv. H O ? 10 mL
1
.5 kb was gel purified using Gen Elute gel extraction kit
2
2
(
Sigma, USA). The purified 16S rRNA gene was cloned
culture supernate) was incubated up to 96 h at 37 °C. 200 lL
of sample was withdrawn at different time intervals (12, 24,
36, 48, 72 and 96 h) and analyzed with HPLC. It was found
that best yield was obtained at 72 h (about 46%) after which
there was no significant increase. For optimization of sub-
strate concentration, initially 1a was added in concentration
into the pGEM-T easy vector system (Promega). The
nucleotide sequencing of the gene was done by using Big
Ò
Dye Terminator Cycle Sequencing Kit (Applied Biosys-
tems) and 3130 9 l Genetic Analyzer (Applied Biosys-
tems). The BLASTN program (http://www.ncbi.nlm.nih.
gov/BLAST/, NCBI, Bethesda, MD) was used for homol-
ogy searches with the standard program default. Multiple
alignments of the sequences were performed using CLUS-
TAL W program [36]. 16S rRNA gene sequence of
P. mandelii KJLPB5 is provided as supplementary data.
-
1
-1
of 1.0 g L and gradually increased up to 3.0 g L
Maximum conversion of 1a to 1b was observed with 2.0 g
.
-
1
L
; yield declining correspondingly with increase of sub-
-
strate concentration to 2.5 g L . No bioconversion was
1
-
1
observed above 3.0 g L . Figure 1 shows the effect of
substrate concentration and varying incubation time at
3
7 °C.
The effect of incubation temperature (20, 28 and 37 °C)
on the oxidation of 4-methoxyphenylpropanol was also
3
Results and Discussions
-
1
3
.1 Screening of Microbial Strains
studied at concentration 2 g L . The conversion increased
from 24 to 46% with the increase of temperature from 20 to
37 °C. Thus, from the results it was ascertained that 2.0 g
Using 4-methoxyphenylpropanol (1a) as a model substrate,
the strains capable of oxidation were rapidly screened
through high performance liquid chromatography (HPLC)
by comparison with reference standard. The products
obtained from bioconversion were confirmed through
GC–MS and NMR. Accordingly, a strain showing good
conversion was selected, subsequently identified by 16S
rDNA sequencing and hereafter named as P. mandelii
KJLPB5 (EMBL # FN811901). The strain is currently
deposited with Institute of Microbial Technology, Chan-
digarh, India.
-
1
L
of substrate concentration, 37 °C of incubation tem-
perature and 72 h of incubation time was optimum for the
conversion.
In our efforts to increase the yield, we shifted our
attention to evaluate the dependence of the above reaction
5
4
0
5
40
3
3
2
2
5
0
5
0
3
.2 Biotransformation Studies with Culture Supernate
of KJLPB5
15
10
5
0
1
0
0 mL of culture supernate of KJLPB5 was taken and
-
.12 mM of 1a (final conc. in medium 2.0 g L ), 30%
1
0
24
48
72
96
H O (6 equiv.) and 0.5 mL [hmim]Br was added to it. The
2 2
Incubation time (h)
reaction mixture was incubated up to 96 h at 37 °C. Bio-
transformation products were analyzed with HPLC. The
pellet constituting of resting cells was also checked for
targeted enzyme activity, however maximum conversion
was observed in culture supernate clearly indicating the
Fig. 1 Effect of substrate concentration and incubation time on the
yield of 1b by P. mandelii KJLPB5. The reactions are performed with
4
-1
-methoxyphenylpropanol as substrate in concentrations (g L ): 1.0
filled diamonds), 2.0 (filled squares), 2.5 (filled triangles) and 3.0
(
(times) at temperature 37 °C in 0.5 mL IL and 6 equiv. of 30% H
2 2
O
1
23

Pseudomonas mandelii KJLPB5 Catalyzed Oxidations in Ionic Liquid
619
on the composition of the ionic liquid (Table 1). We found
that, apart from [hmim]Br, ionic liquids [bmim]Br
Table 2 Oxidation of aryl alcohols/acetates catalyzed by P. mandelii
KJLPB5 and [hmim]Br with H O
2 2
OR
O
(
Table 1, entry 2) and [bmim]Cl (entry 3) were also able to
3
P . m andelii K JLP B 5
[hm im ]B r, 37 oC
provide 1b in more than 40% yield. On the other hand,
acidic ionic liquids like [Hmim]pTSA and [bmim]PF6
provided low yields of 1b (entries 4 and 5) while moderate
conversion (1b in 26% yield) was achieved with
R 1
R 2
R 1
R 2
(a)
(b)
a
Entry
R
1
R
2
2
R
3
Time (h) Yield (%)
[
bmim]BF (entry 6). Interestingly, replacement with basic
4
1
2
3
4
5
6
7
8
9
4-MeOC
6
H
4
C
H
5
5
H
72
72
46
42
35
28
13
ionic liquid [bmim]OH (entry 7) gave only 10% yield of
b. Subsequently, we studied the effect of ionic liquid
3,4-(MeO)
C H
2
C
H
6 3
CH
3
H
1
C H
2
H
72
6
5
amount and oxidant amount on reaction performance. It
was observed that any change in the amount of ionic liquid
or H O led to decrease in product yield (Table 1, entries
4-ClC H
6
CH3
H
72
4
2
6 4
4-NO C H
CH3
H
72
b
16 (59 )
b
12 (57 )
2
2
4-MeOC H
6
4
C H
2
5
COCH3
72
8
–10). We also explored the effectiveness of urea-hydrogen
6 4
4-NO C H
CH3
COCH3
72
2
peroxide (UHP), an adduct of H O , for the above con-
2
2
4-MeOC
6
H
4
H
–
H
–
72
5
version. Significantly lower yield of 1a (entry 11) was
observed along with a number of side products, emphasiz-
ing the benefit of using H O in [hmim]Br. Further, control
Cyclohexanol
120
nd
Reaction conditions: 0.12 mM substrate, 10 mL cell free supernate,
.5 mL IL, 30% H
2
2
0
2 2
O
experiment carried out in absence of [hmim]Br did not
show any conversion into 1b, ascertaining the role of ionic
liquid for above reaction.
a
Product identification with HPLC in comparison to standard and by
b
GC–MS; deacetylated compound as major product
3
.4 Substrate Spectrum
for oxidation with P. mandelii KJLPB5 was 4-methoxy-
phenylpropanol (1a), affording 46% yield with excellent
selectivity. In case of electron withdrawing chloro- (4a)
and nitro- (5a) substitution, lower yields were obtained
(Table 2, entries 4 and 5). When the acetylated 2° aryl
alcohols were subjected to oxidation, corresponding
deacetylated compound was obtained as the major product
To elucidate the scope and structural requirements for
substrates to be converted, a variety of structurally diver-
gent alcohols were subjected to the oxidation under opti-
mized conditions. As shown in Table 2 the best substrate
(
Table 2, entries 6 and 7). On the other hand, 1° aryl
Table 1 Effect of ionic liquid type, ionic liquid amount and con-
on the oxidation of 1a
alcohol (8a) was found to be very less reactive, while
cyclic alcohols (9a) and aliphatic alcohols were not con-
verted at all (Table 2).
centration of H
2
O
2
O
OH
P. mandelii KJLPB 5
30 % H O (6 equiv.)
2 2
These findings suggested the promising use of reaction as
a chemoselective method for oxidizing 2° aryl alcohols in
the presence of 1° aryl alcohols, cyclic and acyclic ones. To
further ascertain this, equimolar mixture of 4-methoxy-
phenylpropanol (1a) and 1° aryl alcohol (Table 3, entry 1)
was treated under developed condition where 1b was
obtained in 46% yield without any significant conversion of
H CO
H CO
Ioni c liquid (0.5 mL),
37 C, 72 h
3
3
o
1b
1
a
a
Entry
Ionic liquid
Yield (%)
1
2
3
4
5
6
7
[hmim]Br
[bmim]Br
[bmim]Cl
46
42
41
6
1
° aryl alcohol (3%). Similarly, oxidation of 1a in presence
of cyclic alcohol (Table 3, entry 2) and aliphatic alcohol
Table 3, entry 3) led to the facile oxidation of only aryl
[Hmim]pTSA
[bmim]PF
[bmim]BF
6
11
26
10
39
18
(
4
alcohol without any conversion or interference by other
alcoholic substrates.
[bmim]OH
[hmim]Br
[hmim]Br
[hmim]Br
[hmim]Br
b
8
c
9
Our quest to increase the yield prompted us to explore
several variants in the above reaction. In one such variant,
instead of adding IL and culture in single step, firstly IL
was added to 1a with constant stirring at 40 °C. After 7 h,
cell free supernate of KJLPB5 was added along with H O
2 2
d
e
1
1
a
0
29
f
1
e
17
Yield on the basis of HPLC conversion with comparison to refer-
b
ence standard; amount of ionic liquid increased from 0.5 to 1 mL;
c
d
amount of ionic liquid decreased from 0.5 to 0.25 mL; amount of
and reaction mixture was incubated for 72 h at 37 °C.
Surprisingly, 4-methoxybenzaldehyde was obtained in 25%
yield along with desired 1a which hinted on the possible
e
increased from 6 to 8 equiv.; multiple products were
3
observed; with urea-hydrogen peroxide (6 equiv.) as oxidant
0% H
2
O
2
f
123

6
20
N. Sharma et al.
2 2
Table 3 Selective oxidation of aryl alcohols catalyzed by P. mandelii KJLPB5 and [hmim]Br with H O
a
b
Entry
1
Alcohols
Time (h)
72
Products
Yield (%)
(46 ? 3)
OH
+
O
CHO
OH
+
3
H CO
3
H CO
H CO
3
H CO
3
2
3
72
72
(43 ? 0)
(45 ? 0)
OH
O
O
OH
+
+
H CO
3
H3CO
OH
O
+
+
OH
O
H3CO
H CO
3
General conditions: 10 mL cell free supernate of P. mandelii KJLPB5, 0.5 mL IL, 6 equiv. of 30% H
a
2
O
2
, 37 °C
b
Equimolar mixture of alcohol; product identification with HPLC in comparison to reference standard and by GC–MS
role of KJLPB5 for oxidative cleavage. Based on the above
observation and our previous finding [30] we envisaged the
direct conversion of aryl alcohols into corresponding one
or two carbon shorter aldehydes employing P. mandelii and
H O in ionic liquid [hmim]Br. For this, in the initial step,
since this area is currently the subject of numerous studies
for development of metal-independent ‘‘green’’ oxidations
[8]. t-anethole was chosen as substrate of choice. Initially,
0.12 mM of the compound was incubated with 10 mL cell-
free supernate of P. mandelii KJLPB5, 0.5 mL [hmim]Br
and 30% H O (6 equiv.) at 37 °C for 72 h resulting in
2
2
a mixture of 4-methoxyphenylpropanol (1a) (0.12 mM)
and [hmim]Br (0.5 mL) was irradiated for 15 min in a
round bottom flask in a focused microwave system
2
2
89% conversion to anisaldehyde (Table 5, entry 1). To rule
out any background activity from IL, control reaction with
[hmim]Br and H O was carried out whereby the yield was
(
150 W, 120 °C), which yielded corresponding dehydrated
2
2
product (confirmed by TLC and HPLC) [30]. In the next
step, 10 mL of culture supernate of KJLPB5 and 6 equiv.
of 30% H O was added to the same pot and reaction
obtained in traces only. Similarly, when the reaction was
carried out without IL, low yield was obtained (about 66%)
hinting at the possible role of IL in activation or stabil-
ization of enzymes [37, 38]. Further, testing of various
alkenes allowed us to elucidate the substrate scope for the
reaction (Table 5). It was clear that the developed system
could not cleave double bond non-conjugated to aromatic
ring, or bulky 1,2-disubstituted alkenes such as trans-
or cis-stilbene (Table 5).
2
2
mixture was incubated for 72 h at 37 °C. To our delight,
various structurally divergent 2° aryl alcohols were suc-
cessfully converted affording quantitative yield (85%) with
excellent selectivity in one pot-two step protocol (Table 4).
Fascinated by this flexible oxidizing ability of KJLPB5,
we sought to exploit the same for oxidative alkene cleavage
Table 5 Oxidative cleavage of aryl alkenes catalyzed by P. mandelii
KJLPB5 and [hmim]Br with H O
2 2
Table 4 Oxidative conversion of 2° aryl alcohols to one or two
carbon shorter benzaldehydes by P. mandelii KJLPB5 with H
2
O
2
O
OR 3
1. [hmim]Br, 15 min M.W.
2. P. mandelii KJLPB5, H2O2, 37 o
O
P. mandelii KJLP B5
[hmim]Br, H2O2, 37 oC
R2
R1
R1
H
R1
H
R1
R2
C
(a)
(b)
(a)
(b)
a
a
Entry
R1
R2
Time (h)
Yield (%)
Entry
R
1
R
2
2
R
3
Time (h) Yield (%)
1
2
3
4
5
6
7
8
4-MeOC
4-MeC
4-ClC
6
H
4
CH
CH
CH
CH
CH
CH
H
3
3
3
3
3
3
72
72
72
72
72
72
72
72
89
52
44
30
36
36
65
nd
1
2
3
4
5
6
8
4-MeOC
6
H
4
C
H
5
H
72
72
72
72
72
72
72
85
77
72
45
36
69
35
3,4-(MeO)
4-MeC
4-ClC
3-NO
4-MeOC
4-BrC
2
C
H
6 3
CH
CH
3
3
H
6
H
4
H
5
H
6
H
4
6
2 6 4
3-NO C H
6
H
4
C
C
C
C
2
H
2
H
2
H
2
H
5
5
5
5
H
6 4
4-BrC H
2
C
6
H
4
H
2-ClC
H
6 4
6
H
4
COCH
H
3
4-MeOC
H
6 4
6 4
H
C
H
6 5
6 5
C H
Reaction conditions: (1) 0.12 mM substrate, 0.5 mL IL, MW for
5 min at 150 W and 120 °C; (2) 10 mL cell free supernate of P.
mandelii KJLPB5, 30% H
1
Reaction conditions: 0.12 mM substrate, 10 mL cell free supernate,
0.5 mL IL, 30% H
O
2 2
2 2
O
a
a
Product identification with HPLC in comparison to standard and by
GC–MS
Product identification with HPLC with comparison to standard and
by GC–MS
1
23

Pseudomonas mandelii KJLPB5 Catalyzed Oxidations in Ionic Liquid
621
3
.5 Effect of Some Inhibitors on Biotransformation
4 Conclusions
In some preliminary experiments, effects of metal chelators
EDTA and EGTA), and sulfhydryl reducing agents (DTT
In summary, a green biocatalytic approach is reported for
the metal-free H O activation using culture supernate of
(
2
2
and Iodoacetamide) on oxidative cleavage reaction, with
t-anethole as model substrate, were studied for an insight
into the identity of components in the supernatant. At
P. mandelii KJLPB5 in neutral IL [hmim]Br for the oxi-
dation of 2° aryl alcohols and sequential synthesis of aryl
aldehydes through alkene cleavage. Although the catalytic
system provided a modest yield for 2° aryl alcohol oxida-
tion, it offers a promising platform for the hitherto
unknown biocatalytic direct synthesis of one or two carbon
shorter aldehydes from 2° aryl alcohols. Further efforts for
enhancement of yield and substrate scope are in progress.
5
5
mM concentration of each supplement used, more than
0% reduction in the product yield was observed with
metal chelators. Similarly, significant inhibition was
caused by Iodoacetamide resulting in very low product
yield (\20%) however, DTT (Dithiothreitol) has no
inhibitory effect on the transformation.
Acknowledgments NS and UKS are indebted to CSIR New Delhi
for the award of research fellowships. CSIR Delhi is acknowledged
for financial support (NWP0006, MLP0025 and MLP0034). The
authors are grateful to the Director, IHBT Palampur, for his kind
cooperation and encouragement.
3
.6 Proposed Mechanism
To demonstrate that above oxidation is biocatalysed, the
transformation of 1a into 1b was carried out with culture
supernate inactivated by heat which resulted in no reaction
at all. Furthermore, neither corresponding epoxide nor diol
was detected during the reaction course leaving out the
possibility of these as intermediates in a possible pathway.
Moreover, the inability of culture supernate to convert
t-anethole epoxide into the corresponding aldehyde hinted
out that the alkene cleavage is a one step mechanism
employing molecular oxygen as oxidant [26]. Further, the
culture supernate was found positive for the catalase
activity. Mechanistically for oxidative-cleavage, it was
presumed that ionic-liquid promotes dehydration through
an initial polarization of the C–O bond by the imidazolium
cation of the ionic liquid [30]. At the same time, molecular
oxygen released by the disproportionation of hydrogen
peroxide by the catalase enzyme present in supernatant
attacks the double bond resulting in formation of corre-
sponding aldehyde via alkene cleavage. The detailed
characterization of reaction mechanism for the above
conversion is under progress.
References
1. Sheldon RA, Kochi JK (1981) Metal catalyzed oxidations of
organic compounds. Academic Press, London
2
. Sheldon RA, Arends IWCE, Ten Brink GJ, Dijksman A (2002)
Acc Chem Res 35:774
. B a¨ umer USt, Sch a¨ fer HJ (2005) J Appl Electrochem 35:1283
3
4. Brooks CD, Huang LC, McCarron M, Johnstone RAW (1999)
Chem Commun 37
5
6
. Liu ST, Reddy KV, Lai RY (2007) Tetrahedron 63:1821
. Larock RC (1999) Comprehensive organic transformations.
Wiley-VCH, New York, pp 1234–1256
7. Beller M, Bolm C (1998) Transition metals for organic synthesis,
vol 2. Wiley-VCH, Weinheim, pp 350–360
8
. Trost BM, Fleming I, Ley SV (1991) Compreshensive organic
synthesis, vol 7. Pergamon, Oxford, pp 251–325
. Hudlicky M (1990) Oxidations in organic chemistry. ACS,
Washington
9
10. Cainelli G, Cardillo G (1984) Chromium oxidants in organic
chemistry. Springer, Berlin
1
1
1. Kon Y, Usui Y, Sato K (2007) Chem Commun 4399
2. Zhang S, Zhao G, Gao S, Xi Z, Xu J (2008) J Mol Catal A Chem
2
89:22
1
1
3. Jiang N, Ragauskas AJ (2005) Tetrahedron Lett 46:3323
4. Hida T, Nogusa H (2009) Tetrahedron 65:270
3
.7 Preparative Scale Evaluation of Oxidative
Biotransformation
15. Ming-Lin G, Hui-Zhen L (2007) Green Chem 9:421
1
1
1
6. Shi F, Tse MK, Beller M (2007) Adv Synth Catal 349:303
7. Muzart J (2003) Tetrahedron 59:5789
8. Velusamy S, Srenvasan A, Punniyamurthy T (2006) Tetrahedron
Lett 47:923
To evaluate the practical use of isolated strain, oxidative
biotransformation was carried out on preparative scale. For
it, five flasks each containing 0.36 mg of 1a was micro-
waved in 1 mL of IL for 15 min and then 30 mL of
P. mandelii KJLPB5 supernate and 6 equiv. H O was
19. Blanchard LA, Hancu D, Beckman EJ, Brennecke JF (1999)
Nature 399:28
2
2
0. Welton T (1999) Chem Rev 99:2071
1. Sharma UK, Sharma N, Kumar R, Kumar R, Sinha AK (2009)
Org Lett 11:4846
2
2
added to it. Flasks were incubated for 72 h at 37 °C.
Product was extracted with ethyl acetate and analyzed with
HPLC where quantitative conversion was confirmed. Col-
umn chromatography of the crude sample over silica gel
with hexane/ethyl acetate (8:2, v/v) gave anisaldehyde with
22. Siebum A, van Wijk A, Schoevaart R, Kieboom T (2006) J Mol
Catal B Enzym 41:141
2
3. Edegger K, Mang H, Faber K, Gross J, Kroutil W (2006) J Mol
Catal A Chem 251:66
4. Kunamneni A, Camarero S, Garc ´ı a-Burgos C, Plou FJ, Ballest-
eros A, Alcalde M (2008) Microb Cell Fact 7:32
2
7
6% yield.
123

6
22
N. Sharma et al.
2
2
2
2
2
3
3
5. Lara M, Mutti FG, Glueck SM, Kroutil W (2008) Eur J Org
Chem 3668
32. Botteghi C, Paganelli S, Moratti F, Marchetti M, Lazzaroni R,
Settambolo R, Piccolo O (2003) J Mol Catal A Chem 200:147
33. Nockemann P, Binnemans K, Driesen K (2005) Chem Phys Lett
415:131
34. Kasana RC, Sharma UK, Sharma N, Sinha AK (2007) Curr
Microbiol 54:457
35. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a
laboratory manual, 2nd edn. Cold Spring Harbor, New York
36. Thompson JD, Higgins DG, Gibson DJ (1994) Nucleic Acid Res
22:4673
37. van Rantwijk F, Sheldon RA (2007) Chem Rev 107:2757
38. Schofer SH, Kaftzik N, Wasserscheid P, Kragl U (2001) Chem
Commun 5:425
6. Mang H, Gross J, Lara M, Goessler C, Schoemaker HE, Guebitz
GM, Kroutil W (2006) Angew Chem Int Ed 45:5201
7. Mang H, Gross J, Lara M, Goessler C, Schoemaker HE, Guebitz
GM, Kroutil W (2007) Tetrahedron 63:3350
8. Sharma A, Kumar R, Sharma N, Kumar V, Sinha AK (2008) Adv
Synth Catal 350:2910
9. Sharma A, Sharma N, Kumar R, Sharma UK, Sinha AK (2009)
Chem Commun 5299
0. Kumar R, Sharma A, Sharma N, Kumar V, Sinha AK (2008) Eur
J Org Chem 5577
1. Lou WY, Chen L, Zhang BB, Smith TJ, Zon MH (2009) BMC
Biotechnol 9:90
1
23