18 - Chapter 10

Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines

10.1 INTRODUCTION
The sonochemical approach is becoming an immensely significant process
intensification tool for conducting organic synthesis (Chebanov et al. 2012; Singh et
al. 2013; Jarag et al. 2011). This technique is not only simple and safe to conduct but
also can assist in preserving green chemistry concepts (Cintas et al. 1999). The
ultrasound energy is derived from cavitation phenomenon involving sequential
formation, growth and collapse of millions of vapour bubbles of microscopic nature
in liquid solution.
This continuous implosive process creates local hot spots in cold fluid with
extremely high temperatures of about 14,000 K and pressure of about 10,000 atm
(Patil et al. 2007). The reactants are thus exposed to such localized conditions
thereby transforming them to highly reactive species for efficient synthesis of
products. The ultrasonic irradiation leads to faster transformations of various
reactions conducted in homogeneous or heterogeneous systems (Carruters et al.
2004). It has been observed that ultrasound, being a mechanical rather than an
electromagnetic wave (Mason et al. 1996), has the ability to augment
biotechnological processes under mild irradiation conditions. One such example
includes enzyme catalyzed organic synthesis where ultrasound is known to increase
the enzymatic activity in comparison to mechanical stirring (Sinisterra et al. 1992).
The reason for the effectiveness of ultrasound is related to the increase in the mass
transfer of the reagents to the enzymatic active site. Ultrasound has also been shown
to increase stability and durability of enzyme biocatalysts (Rokhina et al. 2009).
There are many recent examples reporting combinative use of ultrasound technique
Synthesis of novel colorants for dye-sensitized solar cells and use of greener
protocols for heterocyclic synthesis Page 351

and lipase enzyme in organic synthesis (Konwarh et al. 2012; Batistella et al. 2012;
Gumel et al. 2012; Fiametti et al. 2012).
N-Alkyl aromatic amines are widely used as highly versatile synthetic intermediates
in pharmaceuticals, agrochemicals, bioactive compounds, polymers, and dyes
(Salvatore et al. 2001; Brown et al. 1994). They are also significant pharmacophores
in biologically active compounds in the area of drug discovery (Insaf et al. 1999). In
addition, secondary amines also find application in photography, xerography,
pigments and electronic materials (Wolfe et al. 1998).
These simple but significant moieties have been reportedly synthesized by several
methods including base-mediated N-alkylation using toxic and corrosive alkylating
agents (Larock et al. 1999), reductive amination (Szardenings et al. 1996), direct N-
alkylation by SN2 displacement (Koh et al. 1993), alkylative amination (Alvaro et
al. 2002), use of supported catalysts (Won et al. 2009), metal complexes (Benoit et
al. 2009), etc. However, most of these methods not only use harsh reagents or
catalysts but also lack selectivity giving multiple alkylations, require higher
temperature and longer reaction times. Irradiation techniques like use of microwave
(Marzaro et al. 2009) in aqueous synthesis was also reported but required
temperature as high as 150°C, higher equivalents of alkylating agent and lower
yields in case of electron withdrawing substituents. Ultrasound technique has also
been explored in the past for N-alkylation of weakly nucleophilic amines but used
sodium hydride as a base (Khalaj et al. 2009).
Hence, there is a pressing need to replace these harsh methods with milder one that
would selectively synthesize mono-N-alkyl aromatic amines. In continuation with


our earlier attempts towards the development of environmentally benign process of
mono-N-alkylation of aromatic amines using lipase catalyst (Singh et al. 2011), we
thought of further improving the reaction by exploring the effect of ultrasound
technique. This approach would assist in increasing the enzymatic activity thereby
increasing the rate of reaction. Here, we report, for the first time, the beneficial
application of ultrasound to lipase catalyzed synthesis of mono N-alkyl aromatic
amines in ethanol. Since, the conditions are relatively mild, it is also likely to protect
the enzyme and make it amenable to a possible recycle.

10.2 RESULTS AND DISCUSSION
10.2.1 Variation of organic solvent
We initially investigated the ultrasound-assisted lipase-catalyzed alkylation of
aniline with propyl bromide for optimizing reaction conditions, and the results were
summarized in Table 10.1. The starting materials, aniline and propyl bromide, were
reacted in presence of lipase catalyst using sonication in different organic solvents
such as ethanol, toluene, dichloromethane and hexane. It was demonstrated that
better yield was obtained when the reaction was conducted in ethanol as compared
to any other solvent (entry 1). This indicates that the lipase activity was improved in
ethanol upon irradiation with ultrasound, which could be attributed to its hydrogen
bonding ability that may effectively interact with the enzyme. With regards to
stability of this enzyme in organic solvents, it is known that lipases derived from
Pseudomonas sp. strain are stable in protic polar solvents such as alcohols even after
15 h of interaction with solvent (Pogorevc et al. 2002). The reactions were also
compared in absence of bio-catalyst that resulted in low yields. These observations
suggest that product formation is enhanced by use of lipase catalyst.

Table 10.1: Ultrasound assisted optimization of organic solvent in presence and
absence of lipase catalyst
Entrya Organic solvent Catalyst Yields (%) b
- 30
1. Ethanol
Lipase 85
- 20
2. Toluene
Lipase 65
- 10
3. Dichloromethane
Lipase 30
- -
4. Hexane
Lipase Traces
a
Reaction conditions:
Ultrasonic energy (32 W); aniline (1.0 mL, 10.7 mmol), propyl bromide (1.5 mL, 6.1
mmol), lipase (50 mg, 5 % by weight of amine), organic solvent (6 ml), temp = 30+
2°C, reaction time = 40 min.
b
Isolated yields

10.2.2 Variation of quantity of catalyst
The catalyst loading was varied in N-alkylation of aniline with propyl bromide to
understand the optimum amount of catalyst required for the reaction. It was
observed that the yields improved appreciably for 5% of lipase catalyst by weight of
aromatic amine after which there was not much rise in yield even if the catalyst
quantity was doubled Figure 10.1. It is possible that microscopic agitation through
ultrasound may shift the reaction location to the interface. Hence, under identical US
irradiation, some minimum quantity of enzyme would saturate the interface and any
further increase in the enzyme quantity would not be useful. The shockwaves
produced by the cavitational collapse can create micromixing and facilitate the mass
transfer by increasing the diffusitivity of the large enzyme molecule. This is likely to
promote the movement of the enzyme towards the interface as lipase has natural
affinity for the interface. The facilitation of the enzyme movement towards the
interface could be responsible for the observed enhancement.

Figure 10.1: Ultrasound-assisted optimization of catalyst quantity
a
Reaction conditions: Ultrasonic energy (32 W); aniline (1.0 mL, 10.7 mmol),
propyl bromide (1.5 mL, 16.1 mmol), lipase (% weight by weight of amine),
ethanol (6 ml), temp. = 30+ 2°C, reaction time = 40 min.


10.2.3 Mono-N-alkylation reaction of aniline derivatives with propyl bromide
under varying conditions
In order to understand the significance of ultrasound technique in bio-catalyzed
reaction, the alkylation reaction of various amine derivatives with propyl bromide
was conducted under varying conditions Scheme 10.1. All these optimization
studies were performed in presence of lipase catalyst under both sonochemical as
well as by non-sonochemical method as shown in Table 10.2. It was observed that
ultrasound plays an important role in activating the enzyme catalyst owing to the
fact that after using ultrasound, the results improved considerably both in terms of
reduced reaction time and improved yield. The possible reason for this might be that
ultrasound reduces the diffusional resistance offered to the substrate by the enzyme,
thereby improving the enzyme activity. The non-sonochemical reaction at room
temperature (30+2 °C) in presence of catalyst gave no reaction at all.
Scheme 10.1: Selective N-alkylation of various aromatic amines with propyl
bromide by NUS and US method
Ultrasound CH3
HN
NH2 (30+ 2°C)
Br Lipase Bio-catalyst NUS

+ H3C
Ethanol (30+ 2°C)
NUS R1
R1
(45+ 2°C)
where R 1 = -H, -OCH3, -Cl, -NO2
1 2 NUS - Non-ultrasound
3

Table 10.2: Comparative study of non-ultrasound and ultrasound method in the bio-
catalyzed selective N-alkylation of aromatic amines with propyl bromide
Entrya Products NUSb NUSb USc

(at 30+ 2°C) (at 45+ 2 °C) (at 30+ 2°C)
Reaction Yieldd Reaction Yieldd Reaction Yieldd

Time (%) Time (%) Time (%)
(h) (min.) (min.)
CH3
HN
3a.
4-5 - 110 71 40 85
CH3
HN
3b.
4-5 - 105 76 30 82
CH3
O
CH3
3c. HN
Cl 4-5 - 180 65 45 79
CH3
HN
3d.
4-5 - 240 60 60 69
NO 2
a
Reaction conditions: aromatic amine (1 eq), propyl bromide (1.5 eq), lipase (50mg,
5% weight by weight of amine), ethanol (6 ml)
b
NUS, Non-ultrasound method
c
US, Ultrasound method, (32 W)
d
Isolated yields


10.2.4 Mono-N-alkylation of various aromatic amine substrates with different
alkylating agents
The optimized methodology involving application of ultrasound in lipase catalyzed
alkylation was further extended for other alkyl halides as shown in Scheme 10.2.
Several amine derivatives were reacted with butyl bromide and hexyl bromide. The
results were impressive again as all the mono-N-alkyl products were formed with
improved results Table 10.3. Once again the reactions were continued until no di-N-
alkyl amine formation was observed on thin layer chromatography plate thereby
obtaining maximum monoalkyl compound. The electron donating amines consumed
lesser time for reaction completion as compared to electron withdrawing derivatives
owing to the higher nucleophilicity of the former. In terms of alkylating agents,
although initially, the product formation in propyl derivatives was observed faster
than in hexyl derivatives, however the completion of the reaction took longer time.
However the reaction times and the yields were much improved than that reported in
our earlier published work that used only thermal energy to activate the enzymatic
reaction.
The compounds were characterized by analytical techniques such as FT-IR, 1H-
NMR spectroscopy and mass spectrometry. The relevant peaks of N-H stretching
and bending (above 3000 and 1600- 1650 cm-1 respectively), and C-N stretch were
clearly observed in the FT-IR spectra. In addition, 1H-NMR shows the presence of
alkyl chain protons and aromatic protons. The molecular weight of the compounds
was confirmed by mass spectrometry.

Scheme 10.2: Extension of ultrasound assisted methodology in mono N-alkylation
of aromatic amines with different alkyl bromides using lipase as bio-catalyst
R
NH 2 HN
Lipase Bio-catalyst
+ R-Br
Ultrasound
Ethanol, (30 + 2°C )
R1 R1
4 5 6
where R 1 = -H, -CH 3 , -Cl, -NO 2
R = Butyl, Hexyl
Table 10.3: Ultrasound-assisted mono N-alkylation of aromatic amines with alkyl

bromide by using lipase as bio-catalyst in ethanolic media
Entrya Aromatic Alkyl halide Products Time Yieldsb

amines (min.) (%)
HN CH3
6a. NH2 Butyl bromide 30 75
HN CH3
6b. Hexyl bromide 35 80
HN CH3
18 79
6c. NH2
Butyl bromide
CH3
HN CH3
6d. CH3
Hexyl bromide 24 80
CH3

HN CH3
Cl
6e. NH 2 Butyl bromide 35 70
Cl
6f. HN CH3
Cl
Hexyl bromide 50 75
HN CH3
6g. Butyl bromide 45 65

NH2
NO2
HN CH3
NO 2
6h. Hexyl bromide 70 71
NO 2
a
Reaction conditions: Ultrasound energy (32 W), aromatic amine (1 eq), propyl
bromide (1.5 eq), lipase (50mg, 5% weight by weight of amine), ethanol (6 ml);
temp = (30+ 2°C)
b
Isolated yields

10.2.5 Recycling studies of lipase catalyst
Recycling of lipase catalyst and ethanol was performed for ultrasound-assisted
mono-N-alkylation of aniline with propyl bromide for four successive runs. The
lipase catalyst was easily separable from the reaction mass by filtration and could be
easily recycled. Although the activity was reduced after four runs, the reduction
pattern was similar to the one observed in non-ultrasound method reported earlier
where we assumed that the reduction in yield might be due to inactivation of enzyme
with continuous usage or due to loss of the enzyme during handling. This in turn
indicates that use of ultrasound might not have any significant negative effect on
bio-catalytic activity after many uses. In addition, it is known that the enzyme loses
its activity only when exposed to ultrasound for an extended period (several hours
continuously). Acoustic cavitation phenomena occur over microsecond time scale
and hence, limited exposure to ultrasound is in fact known to activate the enzyme
than deactivate it. The experiments carried out in this work have lasted for maximum
for 70 minutes. Hence, no significant drop in activity (due to ultrasound) is expected
under these conditions. Our earlier work has clearly indicated the fact that short
exposure to ultrasound has no significant effect on the enzyme activity (Save et al.
1994). The results are summarized in graphical representation in Figure 10.2.

Figure 10.2: Recyclability studies of lipase catalyst in ultrasound assisted mono N-

alkylation of aniline with propyl bromide

10.2.6 Plausible mechanism
In accordance with the mechanism of functioning of lipase catalyst in organic
reactions (Save et al. 1994) and with a view of the mechanism reported earlier by us
for selective mono-N-alkylation (Singh et al. 2011), we suggest that lipase plays an
important role in increasing the nucleophilicity of aromatic amines. This could be
possible via proton abstraction of amine by Asp- His dyad as shown in Figure 10.3.
- +
Br R1
H R1 Asp
N O N N H
H
O
His +
R
Asp
Mono N-alkylated product -HBr
O N N H
H
NH
Br
O
Proton abstraction of
Asp
amine by Asp-His dyad
R O N N H
H
O
Figure 10.3: Proposed mechanism for selective mono N-alkylation of aromatic

amines in lipase-catalyzed medium


10.3 EXPERIMENTAL
10.3.1 Materials
The immobilized lipase was acquired from Rossari Bio-tech Pvt. Ltd. It was
obtained from Pseudomonas sp. strain with particle size of less than 60–100 mesh
and activity of 30,000 u/g. FT-IR spectra were recorded on a Bomem Hartmann and
Braun MB-Series FT-IR spectrometer. 1H NMR spectrums were recorded on Varian
300 MHz mercury plus spectrometer and mass spectral data were obtained with a
micromass-Q-TOF (YA105) spectrometer. Common reagent grade chemicals were
procured from M/s S.D. Fine Chemical Ltd, Sigma Aldrich and were used without
further purification.

10.3.2 Ultrasound set-up
Ultrasound for sonochemical synthesis is generated with the help of ultrasonic
instrument set-up (horn type). The specification and details of the set-up, processing
parameters used during the experiments are:
Make: ACE, USA
Operating frequency: 22 kHz
Rated output power: 750 W
Diameter of stainless steel tip of horn:
1.3 × 10-2 m
Surface area of ultrasound irradiating
face: 1.32 × 10-4 m2
Intensity: 3.4 × 105 W/m2.

10.3.3 Lipase-Catalysed N-Alkylation using non-sonochemical (NUS) method
A mixture of aniline (1 mL, 10.7 mmol) and lipase as catalyst (50 mg, 5% by weight
of amine) was taken in ethanol (6 mL). Propyl bromide (1.5 mL, 16 mmol) was
added drop wise to the reaction mixture. The reaction was either continued at room
temperature or at 45 °C (depending on the studies in Table 10.3) until no di-N-alkyl
formation was observed as indicated by thin layer chromatography (TLC). The
reaction time and the respective yields are given in Table 3. The bio-catalyst was
filtered off through a Whatmann-42 filter paper and washed with ethanol. The
filtrate was distilled under vacuum using rotary evaporator to recover ethanol. The
residue was chromatographed on silica gel column and eluted with hexane: ethyl
acetate (9:1), to afford pure alkyl aniline derivatives.
10.3.4 Lipase-Catalysed N-Alkylation using sonochemical (US) method
A mixture of aniline (1 mL, 10.7 mmol) and lipase as catalyst (50 mg, 5% by weight
of amine) was taken in ethanol (6 mL). Alkyl bromide (16 mmol) was added drop
wise under sonication using an ultrasonic horn (ACE horn, 22 kHz frequency) at
40% amplitude with a 5 s ON and 5 s OFF cycle from time of start of addition of
alkyl bromide. The reaction was continued at room temperature until no di-N-alkyl
formation was observed as indicated by thin layer chromatography (TLC). The
reaction time and the respective yields are given in Table 10.1-10.3. The work-up
and catalyst work-up was similar as that described in the above procedure.

10.4 CONCLUSION
In summary, ultrasound was successfully applied in synthesis of mono-N-alkyl
aromatic amines at room temperature in presence of lipase catalyst. The method is
mild, efficient and environmentally benign. Significant improvement in results was
obtained both in terms of yield and reaction times in comparison to non-
sonochemical method. The catalyst and solvent were easily recovered and recycled
for further runs. All these aspects widen the scope of industrial applicability in using
sonochemical energy for selective enzyme-catalyzed reaction.

10.5 Spectral Data of selected compounds
N-Hexyl-4-methylaniline (6d)
λmax (methanol)/nm 238 and 304 ; νmax/cm-1 Above 3000(NH), 1618 (N-H bend),
1367 (C-N), 1519 (C=C); δH( 300MHz; CDCl3;Me4Si) 0.89-1.56 (11H, m, aliphatic
CH), 2.17-2.23 (3H, m, CH3 and NH), 3.2 (3H, s, CH3), 6.50-7.25 ( 5H, m aromatic
CH); m/z (EI) 192.2(M+1);C13H21N calculated m/z: 191.2.
2-Chloro-N-hexylaniline (6f)
λmax (methanol)/nm 238 and 298 ; νmax/cm-1 3445 (NH), 1638 (N-H bend), 1242 (C-
N); δH( 300MHz; CDCl3;Me4Si) 0.90-1.65 (11H, m, aliphatic CH), 3.14 (2H, m,
CH), 4.25(1H, m, NH), 6.62-7.24 ( 4H, m, aromatic CH); m/z (EI)
212.1(M+1);C12H18ClN calculated m/z: 211.1.
N-Hexyl-3-nitroaniline (6h)
λmax (methanol)/nm 381 and 429 ; νmax/cm-1 3411 (NH), 1622 (N-H bend), 1346 (C-
N), 1527 (C=C); δH( 300MHz; CDCl3; Me4Si) 0.90-1.64 (11H, m, aliphatic CH),
3.15 (2H, m, aliphatic CH), 4.04 (1H, b s, NH), 6.88-7.50 (4H, m, aromatic CH);
m/z (EI) 221.1(M+1);C12H18N2O2 calculated m/z: 222.1.

Mass Spectra
HN CH 3
C H3
HN CH 3
Cl

HN CH 3
NO2

1
H- NMR Spectra
HN CH 3
CH3
HN CH 3
Cl

HN CH 3
NO2

FT-IR Spectra
HN CH 3
CH3
HN CH 3
Cl

HN CH 3
NO2

18 - Chapter 10

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

18 - Chapter 10

Uploaded by

Copyright:

Available Formats

Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of

mono-N-alkyl aromatic amines

The sonochemical approach is becoming an immensely significant process

ultrasound energy is derived from cavitation phenomenon involving sequential

formation, growth and collapse of millions of vapour bubbles of microscopic nature

thereby transforming them to highly reactive species for efficient synthesis of

products. The ultrasonic irradiation leads to faster transformations of various

reactions conducted in homogeneous or heterogeneous systems (Carruters et al.

electromagnetic wave (Mason et al. 1996), has the ability to augment

biotechnological processes under mild irradiation conditions. One such example

includes enzyme catalyzed organic synthesis where ultrasound is known to increase

the enzymatic activity in comparison to mechanical stirring (Sinisterra et al. 1992).

to increase stability and durability of enzyme biocatalysts (Rokhina et al. 2009).

Gumel et al. 2012; Fiametti et al. 2012).

in pharmaceuticals, agrochemicals, bioactive compounds, polymers, and dyes

addition, secondary amines also find application in photography, xerography,

pigments and electronic materials (Wolfe et al. 1998).

methods including base-mediated N-alkylation using toxic and corrosive alkylating

alkylation by SN2 displacement (Koh et al. 1993), alkylative amination (Alvaro et

temperature as high as 150°C, higher equivalents of alkylating agent and lower

yields in case of electron withdrawing substituents. Ultrasound technique has also

sodium hydride as a base (Khalaj et al. 2009).

would selectively synthesize mono-N-alkyl aromatic amines. In continuation with

mono-N-alkylation of aromatic amines using lipase catalyst (Singh et al. 2011), we

thought of further improving the reaction by exploring the effect of ultrasound

application of ultrasound to lipase catalyzed synthesis of mono N-alkyl aromatic

the enzyme and make it amenable to a possible recycle.

10.2.1 Variation of organic solvent

We initially investigated the ultrasound-assisted lipase-catalyzed alkylation of

reacted in presence of lipase catalyst using sonication in different organic solvents

such as ethanol, toluene, dichloromethane and hexane. It was demonstrated that

compared in absence of bio-catalyst that resulted in low yields. These observations

suggest that product formation is enhanced by use of lipase catalyst.

Entrya Organic solvent Catalyst Yields (%) b

interface could be responsible for the observed enhancement.

Figure 10.1: Ultrasound-assisted optimization of catalyst quantity

In order to understand the significance of ultrasound technique in bio-catalyzed

studies were performed in presence of lipase catalyst under both sonochemical as

well as by non-sonochemical method as shown in Table 10.2. It was observed that

thereby improving the enzyme activity. The non-sonochemical reaction at room

temperature (30+2 °C) in presence of catalyst gave no reaction at all.

Scheme 10.1: Selective N-alkylation of various aromatic amines with propyl

bromide by NUS and US method

Br Lipase Bio-catalyst NUS

Entrya Products NUSb NUSb USc

Reaction Yieldd Reaction Yieldd Reaction Yieldd

The optimized methodology involving application of ultrasound in lipase catalyzed

obtaining maximum monoalkyl compound. The electron donating amines consumed

lesser time for reaction completion as compared to electron withdrawing derivatives

owing to the higher nucleophilicity of the former. In terms of alkylating agents,

The compounds were characterized by analytical techniques such as FT-IR, 1H-

was confirmed by mass spectrometry.

Table 10.3: Ultrasound-assisted mono N-alkylation of aromatic amines with alkyl

Entrya Aromatic Alkyl halide Products Time Yieldsb

6a. NH2 Butyl bromide 30 75

6b. Hexyl bromide 35 80

6g. Butyl bromide 45 65

Recycling of lipase catalyst and ethanol was performed for ultrasound-assisted

continuously). Acoustic cavitation phenomena occur over microsecond time scale

for 70 minutes. Hence, no significant drop in activity (due to ultrasound) is expected

1994). The results are summarized in graphical representation in Figure 10.2.

Figure 10.2: Recyclability studies of lipase catalyst in ultrasound assisted mono N-

10.2.6 Plausible mechanism