L. Ragupathy et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 270–276
271
below) to the miniemulsion resulting in 6 mg lipase/mL of the total
emulsion. The mixture was stirred at 60 ◦C for 24 h. After 1 day, the
sample was freeze dried for 1–2 days. 1H NMR spectroscopy of the
product was performed to confirm the formation of the product.
There are only a few examples for enzymatic reactions where
the substrate is present in heterophase like lipase catalysis for
synthesizing polyesters [26], optically active amino acids [27],
and monoesters [28] in miniemulsion. It shall be emphasized
that the lipase-catalyzed condensation reactions for synthesizing
monoesters from various linear carboxylic acids and -phenyl
labeled primary alcohols were performed in aqueous miniemul-
sion, i.e., in the presence of large amounts of water. The generated
byproduct water during these condensation reactions at the inter-
face is effectively expelled from the hydrophobic miniemulsion
droplets (considered as separate nanoreactors), which contain the
reactants, into the continuous aqueous phase of the miniemul-
sion. The advantage of using miniemulsions is that stable, small,
and narrowly distributed nanodroplets (30–500 nm) are formed
[28] which provide a large (and stable) interfacial area for the
lipase to catalyze the reaction. Moreover, the miniemulsion pro-
cesses with enzyme catalysis allows the usage of relatively low
amount of surfactants (∼5 wt% with respect to dispersed phase)
compared to the microemulsion process (∼200 wt% with respect
to dispersed phase), which makes easy the product purification
[28–30]. Additionally, by considering the challenges in developing
green chemical technology, the employment of water in miniemul-
sion as a continuous phase and enzyme as a catalyst for performing
organic reactions, would be highly appreciated.
2.3. Simultaneous enzymatic amidation of PD and
15-hydroxypentadecanaoic acid (15-HPD) by OA in miniemulsion
A mixture of 3 mmol of PD, 2 mmol of 15-HPD and 5 mmol of
oleylamine, 50 mg of hexadecane and 5 g of a 1 wt% Lutensol AT50
solution in water was stirred for 1 h at 40 ◦C. Then, the miniemul-
sion was prepared using the procedure as discussed in the previous
section. A suspension of 500 mg of lipase PS from P. cepacia in 2.5 g
of surfactant solution was given to the miniemulsion and stirred at
40 ◦C for 8 days.
2.4. Enzymatic amidation of 15-hydroxypentadecanaoic acid
(15-HPD) by OA in miniemulsion
A mixture of 2.5 mmol of 15-HPD and 2.5 mmol of oleylamine,
50 mg hexadecane, and 5 g of a 1 wt% Lutensol AT50 solution in
water was stirred for 0.5 h at 90 ◦C. Then, the miniemulsion was
prepared by ultrasonicating the mixture for 2 min (30 s pulse with
10 s pause) at 90% amplitude using Branson sonifier W450D, ½ in.
tip. During ultrasonication, a heated oil bath at 90 ◦C was used
instead of ice bath to stop solidification of the reaction mixture.
500 mg of lipase PS from P. cepacia is dispersed in 2.5 g of surfac-
tant solution and given to the miniemulsion and stirred at 40 ◦C
for 48 h. Two more similar miniemulsions were prepared with and
without enzyme, respectively. Directly after preparation they were
freeze dried for 48 h. From the three reaction mixtures, samples
were taken after 12, 24, and 48 h and analyzed by 1H NMR.
In this paper, we are reporting aminolysis reactions of lactones
ambient reaction conditions using lipases as bio-catalysts. The syn-
thesized amide products are entirely new to the chemical world
and might be potentially employed, e.g., as plasticizers and linker
chains for gene delivery applications [31,32]. Kinetic investigations
were performed between PD with OA to picturize the reaction path-
way. The analytical characterization was performed by 1H NMR
spectroscopy and dynamic light scattering (DLS).
2.5. Analytical techniques
Particle sizes were measured in dilute dispersions by means of
dynamic light scattering using a Nicomp particle sizer (model 370,
PSS Santa Barbara, CA, USA) at a fixed scattering angle of 90◦.
1H NMR spectroscopy was performed on a Bruker DRX400 using
either deuterated trifluoroacetic acid (TFA-d1) or deuterated tetra-
chloroethane (C2D2Cl4) as the solvent. The spectra were referenced
to the solvent signal for C2D2Cl4 (ı = 6.00 ppm) while in the case of
TFA-d1 as solvent the spectra were referenced to the signal of the
CH2CH2O protons (ı = 3.94 ppm) of the surfactant Lutensol AT50 to
avoid any trouble with the sensitivity of the signal of the solvent
toward traces of water. 1H NMR analysis in C2D2Cl4 was performed
at 80 ◦C, where the signals corresponding to polyester (4.15 ppm)
and unreacted lactones (4.08 ppm) can be differentiated. However,
1H NMR analysis of any synthesized sample in both of these sol-
vents yields similar results in respect to the calculation of product
conversion.
2. Experimental
2.1. Materials
Dodecylamine, decylamine, octylamine, and hexadecane were
purchased from Aldrich. 16-Hexadecanolide (HD) and 15-
hydroxypentadecanoic acid (15-HPD) were supplied by Alfa Aesar.
15-Pentadecanolide (PD) was procured from Fluka. Oleylamine
(OA) and hexylamine were obtained from Acros. Benzylamine
was purchased from Merck. Lutensol AT50 [poly(ethylene oxide)-
hexadecylether] was kindly donated by BASF. The following
enzymes were used: chirazyme L-5 (candida antarctica) (Roche),
lipase PS (Pseudomonas cepacia), lipase from candida rugosa and
Novozyme 435 (candida antarctica) (all from Amano), Lipase from
pig pancreas and Lipase from Pseudomonas fluorescens (Fluka),
Esterase 009 (recombinant aspergillus oryzae) (Jülich Chiral Solu-
tions GmbH). All chemicals were used without further purification.
Demineralized water was used throughout the experiments.
3. Results and discussion
3.1. Hydrolysis and aminolysis of lactone
2.2. General procedure for the enzymatic aminolysis/hydrolysis of
lactone in miniemulsion
In a first set of experiments, the lipase-catalyzed aminolysis
between two hydrophobic components, PD and OA, was performed
at 60 ◦C for 24 h. For the stabilization of the miniemulsions a
non-ionic surfactant (Lutensol AT50) was chosen to prevent any
eventual denaturing of the enzyme and thus deactivation by the
interaction with a charged surfactant. The amount of surfactant
was always 16 mg/mmol dispersed phase corresponding to 1 wt%
with respect to the continuous aqueous phase.
2.5 mmol of a lactone (PD or HD) and 2.5 mmol of an amine,
50 mg of hexadecane and 5 g of a 1 wt% Lutensol AT50 solution
in water were stirred for 1 h at 60 ◦C for creating emulsions. The
miniemulsion was prepared by ultrasonicating the mixture during
120 s at 90% amplitude using Branson sonifier W450D, ½ in. tip.
From a suspension of 50 mg of lipase PS from P. cepacia in 2.5 g
of surfactant solution was given a certain volume (for details see
The reaction might lead to both, the hydrolysis product, i.e.,
15-HPD and the corresponding amide product, i.e., N-oleyl-15-