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2958 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Weber et al.
carried out at 80 °C in vacuo by reacting 3,3′-thiodipropionic acid,
dimethyl 3,3′-thiodipropionate, 2,2′-thiodiacetic acid, or diethyl 2,2′-
thiodiacetate with long-chain 1-alkanols in the absence of lipase for
several hours.
Enzyme units were calculated from the initial rates (30 min) of
esterification of 3,3′-thiodipropionic acid and 2,2′-thiodiacetic acid as
well as transesterification of dimethyl 3,3′-thiodipropionate and diethyl
2,2′-thiodiacetate with 1-alkanols. The amounts of immobilized lipases
used for the determination of enzyme units were 12.5 mg for the
conversion of 3,3′-thiodipropionic acid and dimethyl 3,3′-thiodipro-
pionate and 50 mg for the conversion of 2,2′-thiodiacetic acid and
diethyl 2,2′-thiodiacetate. One unit of enzyme activity was defined as
the amount of enzyme (g) that produced 1 µmol of dialkyl ester/min.
Values are given as means ( SEM including the number of experiments
(n ) x).
Thin-Layer Chromatography (TLC). Aliquots were withdrawn
from the reaction mixtures, and the conversion was checked by TLC
on 0.3 mm layers of Silica Gel H (VWR International) using iso-hexane-
diethyl ether (7:3, v/v). Free carboxy groups of the reaction products
were methylated by using a solution of diazomethane in diethyl ether.
Spots were located by iodine staining and charring by spraying with
30% sulfuric acid followed by heating (200 °C). The Rf values of the
various compounds were as follows: medium- and long-chain dialkyl
esters of 3,3′-thiodipropionic acid and 2,2′-thiodiacetic acid, 0.6-0.7;
methyl-alkyl esters of 3,3′-thiodipropionic acid and 2,2′-thiodiacetic
acid, 0.40-0.55; dimethyl 3,3′-thiodipropionate and dimethyl 2,2′-
thiodiacetate, 0.30; diethyl 2,2′-thiodiacetate, 0.42; medium- and long-
chain 1-alkanols, 0.2-0.25; and unesterified 3,3′-thiodipropionic acid
and 2,2′-thiodiacetic acid, <0.1. Similarly, 0.5 mm layers of Silica Gel
H were used for the separation of reaction products by preparative TLC.
The various fractions were scraped off the plates and extracted from
silica gel using water-saturated diethyl ether.
Gas Chromatography (GC). In both esterification and transesteri-
fication reactions, aliquots of products were treated with a solution of
diazomethane in diethyl ether to convert the unreacted or hydrolyzed
3,3′-thiodipropionic acid or 2,2′-thiodiacetic acid to the corresponding
methyl esters. The resulting mixture of methyl esters, unreacted
1-alkanols as well as various methyl alkyl- and dialkyl esters of the
above thia-alkanedioic acids, was analyzed by GC. A Hewlett-Packard
(Bo¨blingen, Germany) HP-5890 Series II gas chromatograph equipped
with a flame ionization detector (FID) was used. Separations were
carried out on a 0.1 µm Quadrex 400-1HT (Quadrex Corp., New Haven,
CT) fused silica capillary column, 15 m × 0.25 mm i.d. using hydrogen
as the carrier gas (column pressure, 50 kPa) initially at 60 °C for 2
min, followed by linear programming from 60 to 380 °C at 20 °C ×
min-1 and finally kept at 380 °C for 2 min. Reaction mixtures containing
both dimethyl 3,3′-thiodipropionate and 1-dodecanol were separated
using the following temperature program: 60 °C for 0.5 min, followed
by linear programming from 60 to 110 °C at 8 °C × min-1 (5 min
isothermally), then from 110 to 380 °C at 25 °C × min-1, and finally
kept at 380 °C for 2 min. Injector and detector temperatures were
maintained at 360 and 380 °C, respectively. Reaction mixtures
containing both didodecyl and octylhexadecyl 3,3′-thiodipropionates
were separated on a 0.2 µm DB-23 (J&W, ASS-Chem, Bad Homburg,
Germany) fused silica capillary coulmn, 40 m × 0.18 mm i.d. using
hydrogen as the carrier gas (column pressure, 136 kPa) isothermally
at 250 °C (injector and detector temperature, 300 °C). Peaks in gas
chromatograms were assigned by comparison of their retention times
with those of peaks from TLC fractions, which had been identified by
GC-MS. Response factors of FID were determined using purified
compounds, and percentages of peak areas were calculated using a
Hewlett-Packard GC ChemStation software.
Figure 1. Reaction scheme of the successive lipase-catalyzed esterification
or transesterification of thia-alkanedioates such as 3,3
(n 1; R H) and dimethyl 3,3 -thiodipropionate (n
as well as 2,2 -thiodiacetic acid (n 0; R H) and diethyl 2,2
thiodiacetate (n 0; R ethyl) with 1-hexadecanol yielding, e.g.,
dihexadecyl 3,3 -thiodipropionate and dihexadecyl 2,2 -thiodiacetate.
′
-thiodipropionic acid
)
)
′
)
1; R methyl)
)
′
)
)
′-
)
)
′
′
acid; 3-thiapentan-1,5-dioic acid), and diethyl 2,2′-thiodiacetate as well
as 1-octanol, 1-dodecanol, 1-hexadecanol, and cis-9-octadecen-1-ol were
obtained from Sigma-Aldrich-Fluka (Deisenhofen, Germany). Diethyl
ether, iso-hexane, methyl-tert-butyl ether (MTBE), tert-butyl alcohol,
benzene, acetonitrile, acetone, dichloromethane, sulfuric acid, sodium
carbonate, sodium bicarbonate, sodium sulfate, and potassium hydroxide
were products of VWR International (Darmstadt, Germany). A solution
of diazomethane in diethyl ether was prepared by the reaction of an
etherial solution of N-methyl-N-nitroso-p-toluolsulfonamide (Aldrich)
with potassium hydroxide (8). Immobilized lipase preparations from
Rhizomucor miehei [Lipozyme RM IM, 23 batch interesterification units
(BIU)/g, defined as the amount of enzyme required to incorporate 1
µmol of palmitic acid into trioleoylglycerol/min from an equimolar
mixture at 40 °C; 10% w/w water], Candida antarctica (lipase B,
Novozym 435; 10500 propyl laurate units/g; 2% w/w water), and
Thermomyces lanuginosus [Lipozyme TL IM, 170 interesterification
units novo (IUN)/g] were kindly provided by Novozymes (Bagsvaerd,
Denmark).
Chemical Preparation of Reaction Intermediates. Monododecyl
esters of 3,3′-thiodipropionic acid were prepared by esterification of
3,3′-thiodipropionic acid (891 mg; 5 mmol) and transesterification of
dimethyl 3,3′-thiodipropionate (1031 mg; 5 mmol), respectively, with
1-dodecanol (932 mg; 5 mmol) in the presence of 200 µL of
concentrated sulfuric acid. The best results were obtained using tert-
butyl alcohol as the solvent for the esterification and benzene as the
solvent for the transesterification reaction. The reaction products, i.e.,
3,3′-thiodipropionic acid monododecyl ester and 3,3′-thiodipropionic
acid methyl dodecyl ester (methyl dodecyl 3,3′-thiodipropionate),
respectively, were purified by column chromatography on Silica Gel
60 (VWR International) using mixtures of iso-hexane-diethyl ether and/
or iso-hexane-diethyl ether-acetic acid-water as the eluents.
Lipase-Catalyzed Esterification and Transesterification Reac-
tions. As a typical example, 3,3′-thiodipropionic acid (178 mg, 1 mmol)
was esterified with 1-dodecanol (410 mg, 2.2 mmol) in the presence
of 6-50 mg of the immobilized lipase preparation by magnetic stirring
in a screw-capped tube in vacuo at 80 °C for periods up to 72 h with
water trapping in the gas phase using potassium hydroxide pellets. A
moderate vacuum (80 kPa) was used to prevent substantial loss of
substrates. Samples of the reaction products were withdrawn at various
intervals, extracted with MTBE at 50 °C, and filtered through a 1 µm
syringe filter to separate the biocatalyst. An aliquot of the filtrate was
analyzed as given below. Similarly, 2,2′-thiodiacetic acid (150 mg, 1
mmol) was esterified with 1-hexadecanol (2.2 mmol). Dimethyl 3,3′-
thiodipropionate (206 mg) and diethyl 2,2′-thiodiacetate (208 mg), 1
mmol each, were transesterified with 1-alkanols (2.2 mmol) under
identical conditions as described above for esterification reaction using
various lipases (6-50 mg, each) as biocatalysts. To determine the chain-
length specificity of Novozym 435, 3,3′-thiodipropionic acid was
esterified with an equimolar mixture of 1-octanol, 1-dodecanol, and
1-hexadecanol under the conditions described above using 12.5 mg of
the biocatalyst and a reaction time of 15 min. Blank experiments were
High-Performance Liquid Chromatography (HPLC). Aliquots
of thia-alkanedioic acid dialkyl esters were analyzed for their composi-
tion by HPLC as follows. The HPLC system consisted of a Merck-
Hitachi pump L-6200 (E. Merck) equipped with a column oven (VDS
Optilab, Berlin, Germany) set at 25 °C, a UV/vis HPLC 332 detector
(E. Merck) set to a wavelength of 210 nm, and a PL-ELS 2100 (Polymer
Laboratories, Darmstadt, Germany) ELSD detector (thermostated to
30 °C for acetonitrile and 40 °C for mixtures of acetonitrile-acetone),
which were used in series. Mass and UV traces were monitored and