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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
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
2004). It has been observed that ultrasound, being a mechanical rather than an
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
There are many recent examples reporting combinative use of ultrasound technique
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
and lipase enzyme in organic synthesis (Konwarh et al. 2012; Batistella et al. 2012;
N-Alkyl aromatic amines are widely used as highly versatile synthetic intermediates
(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
These simple but significant moieties have been reportedly synthesized by several
agents (Larock et al. 1999), reductive amination (Szardenings et al. 1996), direct N-
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
been explored in the past for N-alkylation of weakly nucleophilic amines but used
Hence, there is a pressing need to replace these harsh methods with milder one that
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
amines in ethanol. Since, the conditions are relatively mild, it is also likely to protect
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
10.2 RESULTS AND DISCUSSION
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
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
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protocols for heterocyclic synthesis Page 354
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
Table 10.1: Ultrasound assisted optimization of organic solvent in presence and
absence of lipase catalyst
- 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
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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
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protocols for heterocyclic synthesis Page 356
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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.
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protocols for heterocyclic synthesis Page 357
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
10.2.3 Mono-N-alkylation reaction of aniline derivatives with propyl bromide
under varying conditions
reaction, the alkylation reaction of various amine derivatives with propyl bromide
was conducted under varying conditions Scheme 10.1. All these optimization
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,
Ultrasound CH3
HN
NH2 (30+ 2°C)
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
Table 10.2: Comparative study of non-ultrasound and ultrasound method in the bio-
catalyzed selective N-alkylation of aromatic amines with propyl bromide
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
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
10.2.4 Mono-N-alkylation of various aromatic amine substrates with different
alkylating agents
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
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.
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
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protocols for heterocyclic synthesis Page 360
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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
HN CH3
HN CH3
18 79
6c. NH2
Butyl bromide
CH3
HN CH3
6d. CH3
Hexyl bromide 24 80
CH3
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protocols for heterocyclic synthesis Page 361
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
HN CH3
Cl
6e. NH 2 Butyl bromide 35 70
Cl
6f. HN CH3
Cl
Hexyl bromide 50 75
HN CH3
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
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protocols for heterocyclic synthesis Page 362
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
10.2.5 Recycling studies of lipase catalyst
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
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
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.
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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protocols for heterocyclic synthesis Page 364
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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
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
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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
300 MHz mercury plus spectrometer and mass spectral data were obtained with a
procured from M/s S.D. Fine Chemical Ltd, Sigma Aldrich and were used without
further purification.
10.3.2 Ultrasound set-up
instrument set-up (horn type). The specification and details of the set-up, processing
1.3 × 10-2 m
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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
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
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
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.
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
10.4 CONCLUSION
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
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protocols for heterocyclic synthesis Page 368
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
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
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,
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);
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
Mass Spectra
HN CH 3
C H3
HN CH 3
Cl
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
HN CH 3
NO2
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
1
H- NMR Spectra
HN CH 3
CH3
HN CH 3
Cl
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protocols for heterocyclic synthesis Page 372
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
HN CH 3
NO2
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Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
FT-IR Spectra
HN CH 3
CH3
HN CH 3
Cl
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protocols for heterocyclic synthesis Page 374
Chapter 10: Ultrasound-assisted intensification of biocatalyzed synthesis of
mono-N-alkyl aromatic amines
HN CH 3
NO2
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