Mapping the hydrolytic and synthetic selectivity of a type C feruloyl esterase (StFaeC) from Sporotrichum thermophile using alkyl ferulates

Article abstract of DOI:10.1016/j.tetasy.2004.11.037

The active site of Sporotrichum thermophile type C feruloyl esterase (StFaeC) was probed using a series of C₁-C₄ alkyl ferulates. The affinities for straight and branched alkyl ferulates were demonstrated by the K_m values of 1.64-0.51 and 0.19-0.1, respectively. Comparison of k_cat and k_cat/K_m values shows that the enzyme hydrolyzed n-propyl ferulate faster and iso-propyl ferulate more efficiently. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. Lengthening the aliphatic side chain was the most significant factor causing lower synthetic activity of the enzyme. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic process, such as the temperature and substrate concentration have been investigated. Under identical reaction conditions, the enzyme feruloylated other monosaccharides such as d-arabinose, d-glucose, d-xylose, d-mannose, d-fructose, d-galactose, d-ribose and model substrates such as 4-nitrophenyl α-l-arabinofuranoside and 4-nitrophenyl α-l-arabinopyranoside.

Full text of DOI:10.1016/j.tetasy.2004.11.037

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 373–379
Mapping the hydrolytic and synthetic selectivity of a type C
feruloyl esterase (StFaeC) from Sporotrichum thermophile
using alkyl ferulates
Christina Vafiadi,a Evangelos Topakas,a Ken K. Y. Wong,b
Ian D. Sucklingb and Paul Christakopoulosa,*
aBiotechnology Laboratory, Chemical Engineering Department, National Technical University of Athens,
5 Iroon PolytechniouStr, ZografouCampus, 15700 Athens, Greece
bEnsis Papro, Forest Research, Private Bag 3020, Rotorua, New Zealand
Received 2 November 2004; accepted 18 November 2004
Available online 23 December 2004
Abstract—The active site of Sporotrichum thermophile type C feruloyl esterase (StFaeC) was probed using a series of C1–C4 alkyl
ferulates. The affinities for straight and branched alkyl ferulates were demonstrated by the Km values of 1.64–0.51 and 0.19–0.1,
respectively. Comparison of kcat and kcat/Km values shows that the enzyme hydrolyzed n-propyl ferulate faster and iso-propyl
ferulate more efficiently. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl
group transfer to L-arabinose, using as a reaction system a ternary water–organic mixture consisting of n-hexane, t-butanol and
water. Lengthening the aliphatic side chain was the most significant factor causing lower synthetic activity of the enzyme. The reac-
tion parameters affecting the feruloylation rate and the conversion of the enzymatic process, such as the temperature and substrate
concentration have been investigated. Under identical reaction conditions, the enzyme feruloylated other monosaccharides such as
D-arabinose, D-glucose, D-xylose, D-mannose, D-fructose, D-galactose, D-ribose and model substrates such as 4-nitrophenyl a-L-
arabinofuranoside and 4-nitrophenyl a-L-arabinopyranoside.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
In spite of numerous reports on esterases and lipases
acting as biosynthetic catalysts, enzymatic esterification
of phenolic acids has been rarely reported, as in general,
enzymatic esterification offers an alternative to the poor
selectivity of chemical synthesis.9 Recently, the potential
use of ternary water–organic solvent mixtures,5,6,10 or
oil-in-water microemulsions11 as a reaction system for
the esterification or transesterification of various pheno-
lic acids catalyzed by feruloyl esterases has been re-
ported. The direct esterification of phenolic acids with
aliphatic alcohols catalyzed by various lipases in organic
media has also been reported, but the reaction rate and
yield was low.12–16
Over the last 10 years, feruloyl esterases (FAE, E.C.
3.1.1.73) responsible for cleaving the ester link between
the polysaccharide main chain of xylans and monomeric
or dimeric ferulic acid have been purified and partially
characterized.1–6 These enzymes act synergistically with
xylanases to hydrolyze ester-linked ferulic acid (FA)
from cell wall material.5–7 Feruloyl esterases have now
been classified into four types (A–D) based on their
specificity towards mono- and di-ferulates, for substitu-
tions on the phenolic ring, and on their amino acid
sequence identity.8 The nomenclature of feruloyl
esterases follows both the source of the enzyme and
the type of feruloyl esterases (e.g., the type C feruloyl
esterase from Sporotrichum thermophile is termed
StFaeC).
It has been reported that the synthetic activity pattern
of feruloyl esterases for the transesterification of vari-
ous methyl esters of cinnamic acids is similar to that
of their hydrolytic action.6,10,17 The active sites of feru-
loyl esterases from mesophilic and thermophilic sources
were probed using methyl esters of phenylalkanoic
acids.18 Type B feruloyl esterases were found to be
*
Corresponding author. Tel.: +30 210 7723231; fax: +30 210
7723163; e-mail: hristako@orfeas.chemeng.ntua.gr
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.037

374
C. Vafiadi et al. / Tetrahedron: Asymmetry 16 (2005) 373–379
H
O
H3CO
O
HO
H
OH
O
O
H
H
O
OH
HO
H
OH
H3
C
OH
+
+
HO
OH
H
OCH3
OCH3
OH
HO
Scheme 1. Transesterification of methyl ferulate with L-arabinose.
appropriate biocatalysts for the synthesis of hydroxyl-
ated phenolic compounds while type A feruloyl esterases
are suitable for the synthesis of methoxylated phenolic
compounds. The type C feruloyl esterase StFaeC
demonstrated maximum hydrolytic activity against 4-
hydroxy-3-methoxy cinnamate, indicating that it may
be the most promising type of feruloyl esterase as a bio-
catalyst for the enzymatic feruloylation of aliphatic
alcohols and oligo- or polysaccharides. Recently, it
was reported that StFaeC catalyzed the transfer of the
feruloyl group to L-arabinose (Scheme 1) in a ternary
water–organic mixture consisting of n-hexane, t-butanol
and water system, with about a 40% conversion of L-
arabinose to its feruloylated derivative was achieved.17
This work was the first example of esterification of a
sugar with unsaturated arylaliphatic acids like methoxy-
lated or hydroxylated derivatives of cinnamic acids
(such as ferulic acid). Lipases are not able to catalyze
such a reaction due to an electronic and/or steric
effect.12,16,19
2.2. Hydrolytic and synthetic specificity of feruloyl
esterase for alkyl ferulates
The feruloyl esterase was tested first for activity against
a range of alkyl ferulates (Table 1). A series of seven
alkyl ferulates were tested as substrates in order to deter-
mine the specificity of the feruloyl esterase for straight
and branched C1–C4 aliphatic chains. The affinities for
straight and branched alkyl ferulates were demonstrated
by the Km values of 1.64–0.51 and 0.19–0.1, respectively.
The comparison of kcat and kcat/Km values shows that
the enzyme hydrolyzed faster n-propyl ferulate and
more efficiently iso-propyl ferulate.
Comparison of the specificity profiles obtained for
transesterification with those for hydrolysis shows nota-
ble differences. StFaeC shows strong preference for
short chain length alkyl ferulates and especially for
methyl ferulate. It has been reported that the synthetic
activity pattern of StFaeC for the transesterification of
various methyl esters of cinnamic acids is similar to that
of their hydrolytic action.17 The type C feruloyl esterase
StFaeC demonstrated maximum hydrolytic activity
against ferulate esters. Maintaining the ferulate struc-
ture but altering the alkyl substitutions of the carboxyl
group the esterase demonstrated maximum synthetic
activity against the methyl ester showing the require-
ment of short alkyl chain length for the effective esterifi-
cation of L-arabinose. It seems that increasing the steric
congestion around the ester group by having a more ste-
rically demanding alkyl group increases both the affinity
for the alkyl ferulate and the efficiency of the hydrolysis.
This is in agreement with the fact that feruloylated car-
bohydrates such as 4-nitrophenyl 5-O-trans-feruloyl-a-
L-arabinofuranoside, which is sterically of similar size
to those obtained with iso-propyl and iso-butyl esters
hydrolyzed by StFaeC 11 times more efficiently than
methyl ferulate.17 Other feruloyl esterases have been
shown to de-esterify ferulic acid at a greater efficiency
if it is attached to a sugar than a short alcohol,23 and
the catalytic efficiency of the hydrolysis of 5,50-diferulic
acid by AnFaeA was 20-fold greater when the diferulate
was ester linked to sugars than ethyl groups.24,25
Phenolic acid sugar esters have demonstratable anti-
tumoric activity and have the potential to be used to
formulate antimicrobial, antiviral and/or anti-inflamma-
tory agents.20–22 As esters based on unsaturated arylali-
phatic acids, such as cinnamic acid and its derivatives,
are known to display anticancer activity,19 specific feru-
loyl esterases could be employed in the tailored synthesis
of such pharmaceuticals.
In the present study, factors including reaction temper-
ature, and the type of alkyl ferulates used as feruloyl
donors were investigated to evaluate their effect on
initial rate and conversion of feruloyl arabinose.
Furthermore, the correlation between hydrolytic and
synthetic activity of the esterase against different types
of straight and branched C1–C4 alkyl ferulates has been
investigated.
2. Results and discussion
2.1. Choice of solvents
The enzyme synthetic activity was tested in a n-hexane/t-
butanol/water (53.4:43.4:3.2 v/v/v) ternary system moni-
toring the transesterification of ferulic acid methyl ester
with L-arabinose. This condition was found to be the
best and the synthetic reaction reached 40% conversion
after 160 h.18
2.3. Effect of temperature
In equilibrium-controlled synthesis, the reaction temper-
ature is the most critical factor to determine the conver-
sion. As shown in Figure 1, the initial rates increased
with the increase of temperature up to 45 ꢁC, while

C. Vafiadi et al. / Tetrahedron: Asymmetry 16 (2005) 373–379
Table 1. Specificity of feruloyl esterase (StFaeC) for alkyl ferulates
375
Structure
Name
Hydrolysis
Transesterification conversion (%)
O
O
Km
1.64 (0.10)
156 (5)
95 (7)
kcat
kcat/Km
Methyl ferulate
30.0
OCH3
OH
O
O
Ethyl ferulate
Km
0.51 (0.05)
88 (4)
171 (13)
6.3
kcat
kcat/Km
OCH3
OH
O
O
n-Propyl ferulate
Km
0.51 (0.04)
326 (12)
636 (59)
3.8
kcat
kcat/Km
OCH3
OH
O
O
n-Butyl ferulate
Km
0.73 (0.05)
260 (22)
357 (23)
3.4
kcat
kcat/Km
OCH3
OH
O
O
iso-Propyl ferulate
Km
0.1 (0.01)
99 (2)
1016 (96)
3.3
kcat
kcat/Km
OCH3
OH
O
O
2-Butyl ferulate
Km
0.16 (0.01)
53 (1)
328 (24)
2.7
kcat
kcat/Km
OCH3
OH
O
O
iso-Butyl ferulate
Km
kcat
0.19 (0.01)
96 (2)
450 (34)
4.2
kcat/Km
OCH3
OH
Kinetic constants (Km and kcat) were determined using GraFit program module, which also gives an estimate of the standard error (numbers in
parentheses). Km is expressed as mM and kcat as kat/mol enzyme.
optimum conversion was achieved at 35 ꢁC. The esterase
exhibits highest hydrolytic activity around 55 ꢁC, but at
temperatures above 45 ꢁC enzyme deactivation also
takes place after 6 h of incubation Figure 2.

376
C. Vafiadi et al. / Tetrahedron: Asymmetry 16 (2005) 373–379
Figure 3. Effect of methyl ferulate concentration on initial rate (d) and
conversion (s). The reactions were performed in n-hexane/t-butanol/
water (53.4:43.4:3.2 v/v/v) ternary system, 30 mM L-arabinose and
0.037 nM enzyme at 35 ꢁC for five days.
Figure 1. Effect of temperature on initial rate (d) and conversion (s).
The reactions were performed in n-hexane/t-butanol/water
(53.4:43.4:3.2 v/v/v) ternary system using 100 mM methyl ferulate,
30 mM L-arabinose and 0.037 nM enzyme for five days.
Figure 4. Effect of L-arabinose concentration on initial rate (d) and
conversion (s). The reactions were performed in n-hexane/t-butanol/
water (53.4:43.4:3.2 v/v/v) ternary system using 100 mM methyl
ferulate, and 0.037 nM enzyme at 35 ꢁC for five days.
Figure 2. Effect of temperature on hydrolytic activity (d) and stability
(s). For stability, the enzyme solution in pH 6 was incubated for 6 h at
various temperatures, and then the residual enzyme activities were
assayed. For activity, the enzyme activity was assayed at various
temperatures by the standard assay method.
2.5. Structural characterization of feruloyl transfer
products on L-arabinose
2.4. Effect of substrate concentration on the transesteri-
fication reaction
The feruloylated L-arabinose formed was isolated by
preparative TLC and identified by 1H NMR. The chem-
ical shifts were similar to those published for 5-O-(trans-
feruloyl)-L-arabinofuranose isolated from wild rice
(Zizania aquatica).26 In agreement with the assigned
structure, FTIR analysis of the purified product indi-
cated the presence of an ester band at 1725 cmÀ1 and
analysis by electrospray mass spectrometry operating
in the negative ion mode gave an [MÀ1] peak at m/z 325.
Under the reaction conditions (35 ꢁC, esterase 0.037 nM;
methyl ferulate as feruloyl donor) the effect of L-arabi-
nose and methyl ferulate concentration on the L-arabi-
nose feruloylation rate was studied. The dependence of
the initial rates (Figs. 3 and 4) as a function of the sub-
strate concentrations were used to calculate apparent
Km values for both substrates. As it can be seen from
Figures 2 and 3 the feruloylation reaction follows
Michaelis–Menten kinetics. The calculated apparent
Km and Vmax values for L-arabinose and methyl ferulate
2.6. Feruloylation of other carbohydrates
Results of screening for different carbohydrate feruloyl
acceptors showed that the enzyme had broad acceptor
specificity (Fig. 5). The saccharides having either a pyra-
nose or a furanose ring in their chemical structure are
are: Km ( -arabinose) = 33.2 mM, Km
L
=
(methyl ferulate)
68.9 mM, Vmax ( -arabinose) = 28.2 mmol LÀ1 hÀ1 mgÀ1
,
L
Vmax (methyl ferulate) = 45.5 mmol LÀ1 hÀ1 mgÀ1
.

C. Vafiadi et al. / Tetrahedron: Asymmetry 16 (2005) 373–379
377
Figure 5. S. thermophile feruloyl esterase StFAEC catalyzed feruloylation of various monosaccharides (A) and p-nitrophenyl glycosides of arabinose
(B) in n-hexane/t-butanol/water (53.4:43.4:3.2 v/v/v) using methyl ferulate as feruloyl donor. (A) Monosaccharides after five days incubation with
0.037 nM StFAEC: L-arabinose (1); D-arabinose (2); D-glucose (3); D-xylose (4); D-mannose (5); D-fructose (6); D-galactose (7); D-ribose (8). (I)
Carbohydrates, (II) feruloylated carbohydrates. (B) p-Nitrophenyl arabinopyranoside (1); p-nitrophenyl arabinopyranoside after five days
incubation with 0.037 nM StFAEC (2); p-nitrophenyl arabinofuranoside (3); p-nitrophenyl arabinofuranoside after five days incubation with
0.037 nM StFAEC (4); sugar components were detected on dried chromatograms by the aniline–hydrogen phthalate reagent (A) and ethyl acetate–
benzene–iso-propanol reagent (B).
acceptors and this was further confirmed by the feruloyl-
ation of both 4-nitrophenyl a-L-arabinofuranoside and
4-nitrophenyl a-L-arabinopyranoside. Furthermore, ste-
reoselectivity for feruloylation of D- and L-enantiomers
of arabinose was not observed. A protease from Strepto-
myces sp. also esterified D- and L-enantiomers of arabi-
nose to synthesize vinyl arabinose esters but enzymatic
transesterification proceeded without transesterification
of arabinopyranose.27 It was reported that oligosaccha-
rides containing C-5 modified D-arabinofuranosyl resi-
dues such as the D-feruloylated arabinose, are of
interest as potential inhibitors of the a-(1!5)-arabino-
syltransferase involved in the assembly of mycobacterial
cell-wall arabinan.28,29 Mycobacterial infections and
most notably tuberculosis has long been a cause of mor-
bidity and mortality worldwide. Over the past two dec-
ades, there has been an increased interest in developing
new drugs to fight this deadly disease.
measured by HPLC as previously described.18 All assays
were prepared and analyzed in duplicate, with <10%
standard error for each set of results. The amount of free
acid released was quantified against standard curves.
One unit of activity (1 U) is defined as the amount of en-
zyme (mg) releasing 1 lmol of free acid per minute
under the defined conditions. The relative molecular mass
for the feruloyl esterase StFaeC was 46 kDa (homo-
dimers of 23 kDa).17 Kinetic constants (kcat, Km) were
determined from Michaelis–Menten equation, using
Lineweaver–Burk double reciprocal plots. Values were
estimated using a non-linear regression model (GraFit)
that also gives an estimate of the standard error of each
parameter.31
The effect of temperature on enzyme hydrolytic activity
and stability was measured on methyl ferulate (MFA;
2 mM) using HPLC for detection of FA. The optimum
temperature was determined by assaying the enzyme
activity at various temperatures (30–70 ꢁC) for 15 min
in 0.1 M MOPS buffer, pH 6.0. The thermostability
was determined by measuring the residual activity at
50 ꢁC and pH 6.0, after incubation of the purified ester-
ase between 30 and 70 ꢁC and pH 6.0, for 6 h.
From these results, the esterification of a broader range
of carbohydrates seems reasonable and feruloyl ester-
ases could be employed in the tailored synthesis of phe-
nolic sugar esters. There has been a recent report on
starch chemically esterified with ferulic acid (starch feru-
late).30 This polymer showed lower viscosity, higher
water-holding capacity and much less retrogradation
during low temperature storage compared to native
maize starch. The authors found that starch ferulate
served as a better source of ferulic acid than wheat bran
(dietary fibre) in the colon, where it was degraded by
microbiotica. The potential of using feruloyl esterases
for the synthesis of feruloylated oligomers or polymers
opens the door to design probiotics and modified bio-
polymers with altered properties and bioactivities.
3.2. Reaction solution
The surfactantless microemulsions were prepared by
mixing n-hexane, t-butanol and 20 mM buffer piper-
azine–HCl pH 6.0 in the required volumes, followed
by vigorous shaking for several seconds until a stable
transparent solution was obtained. Alkyl ferulates were
diluted in the mixture of n-hexane and t-butanol.
Enzyme, L-arabinose and other carbohydrates were
introduced in the form of concentrated stock solution
in buffer. Enzymic transesterification was carried out
in sealed flask at different temperatures without stirring.
3. Experimental
3.1. Enzyme
Aliquots (5 lL) of reaction mixtures were spotted on
aluminium sheets coated with Silica gel 60 (Merck, Ger-
many). The solvent system that was used for the resolu-
tion of the feruloylated product formed by the
transesterification of purified StFaeC was chloroform/
methanol/water (65:15:2, v/v/v). TLC plates were visual-
ized under a UV lamp. Sugar components were detected
The esterase described here was purified to homogeneity
from culture supernatants of S. thermophile grown on
wheat straw, as described previously.17 The esterase
activities were assayed using methyl esters of various
alkyl ferulates (Table 1) and the release of ferulic acid

378
C. Vafiadi et al. / Tetrahedron: Asymmetry 16 (2005) 373–379
on dried chromatograms by the aniline–hydrogen phtha-
late reagent.32 Transesterification of 4-nitrophenyl a-L-
arabinofuranoside and 4-nitrophenyl a-L-arabinopyr-
anoside was examined in the solvent system ethyl ace-
tate/benzene/iso-propanol (2:1:0.1, v/v/v). TLC plates
were visualized under a UV lamp. Sugar components
were detected on dried chromatograms by N-(1-naph-
thyl)ethylenediamine dihydrochloride reagent.33
developed using mixtures of methanol and water con-
taining 0.1% H3PO4 and visualized using UV fluores-
cence quenching at 254 nm.
The alcohol was removed under reduced pressure. The
product was dissolved in dichloromethane (120 mL),
washed with saturated aqueous NaHCO3 (3 · 150 mL)
and saturated aqueous NaCl (150 mL), dried (MgSO4)
and concentrated under reduced pressure to afford the
crude ester (95–97%). The esters were recrystallized from
ethyl acetate/hexanes. Proton and 13C NMR spectro-
scopy was done in CDCl3 with a Bruker DRX 400 spec-
trometer, equipped with a 5 mm 1H/13C dual inverse
broad probe at 400.13 MHz (1H) and 100.62 MHz (13C).
Qualitative analysis of samples was made by HPLC on a
C18 Nucleosil column (250 mm · 4.6 mm) (Macherey
Nagel, Dren, Germany). Detection was achieved by a
Jasco UV-975 detector set at 300 nm based on calibra-
tion curve prepared using standard solutions of the
product in water/methanol 50:50 (v/v). The reaction
mixture (100 lL), was diluted with a solution of water/
methanol 50:50 (v/v) (100–1000 lL) before analysis. Elu-
tion was conducted with acetonitrile/water/formic acid
(1.5:7:1) as mobile phase at a flow rate of 1.0 mL minÀ1
and at ambient temperature. Yields for the synthesis of
phenolic sugar ester were calculated from the amount of
L-arabinose having reacted compared to the initial
quantity of the sugar. No L-arabinose consumption
was observed in the absence of esterase preparation.
3.4.1. Ethyl ferulate. 1H NMR, d, ppm: 1.32 (3H, t,
J = 7.0 Hz, CH2CH3), 3.91 (3H, s, OCH3), 4.25 (2H,
q, J = 7.0 Hz, CH2CH3), 5.93 (1H, s, OH), 6.27 (1H,
d, J = 15.9 Hz, CHCHCOO), 6.89–7.07 (3H, m, aro-
matic), 7.60 (1H, d, J = 15.9 Hz, CHCHCOO). 13C
NMR, d, ppm: 14.4 (CH2CH3), 55.9 (OCH3), 60.3
(CH2CH3), 109.3–147.9 (aromatic and CHCHCOO),
167.3 (CHCHCOO).
3.4.2. n-Propyl ferulate. 1H NMR, d, ppm: 0.99 (3H, t,
J = 7.4 Hz, CH2CH2CH3), 1.72 (2H, m, CH2CH2CH3),
3.89 (3H, s, OCH3), 4.15 (2H, t, J = 6.7 Hz,
CH2CH2CH3), 6.14 (1H, s, OH), 6.29 (1H, d,
J = 15.9 Hz, CHCHCOO), 6.90–7.06 (3H, m, aromatic),
7.60 (1H, d, J = 15.9 Hz, CHCHCOO). 13C NMR, d,
ppm: 10.8 (CH2CH2CH3), 22.4 (CH2CH2CH3), 56.2
(OCH3), 66.4 (CH2CH2CH3) 109.8–148.3 (aromatic
and CHCHCOO), 167.8 (CHCHCOO).
The enzymatic synthetic reactions were performed with
0.037 nM StFaeC.
3.3. Structural characterization of phenolic sugar ester
Proton NMR spectroscopy was performed in CDCl3
with a Bruker DRX 400 spectrometer, equipped with a
1
5 mm H/13C dual inverse broad probe at 400.13 MHz.
Mass spectrometry was done using an Agilent 1100
MSD ion trap. The L-arabinose and the reaction prod-
uct were dissolved in 50/50 methanol/water with 0.2%
formic acid. The mass spectrum of L-arabinose is ob-
tained in positive mode and the mass spectrum of the
reaction product is collected in negative mode.
3.4.3. iso-Propyl ferulate. 1H NMR, d, ppm: 1.29 (6H,
d, J = 6.1 Hz, CH(CH3)2), 3.86 (3H, s, OCH3), 5.12 (1H,
septet, J = 6.1 Hz, CH(CH3)2), 6.25 (1H, d, J = 16.0 Hz,
CHCHCOO), 6.29 (1H, s, OH), 6.88–7.04 (3H, m, aro-
matic), 7.58 (1H, d, J = 16.0 Hz, CHCHCOO). 13C
NMR, d, ppm: 22.3 (CH(CH3)2), 56.2 (OCH3), 68.0
(CH(CH3)2), 109.8–148.3 (aromatic and CHCHCOO),
167.3 (CHCHCOO).
FTIR (Nicolet Magna-IR 560) analysis of reaction
product was performed in order to detect the ester bond.
FTIR spectra were obtained at 4 cmÀ1 resolution with
200 scans. The spectrometer was equipped with a DTGS
KBr detector and a DRIFT accessory. The solid sam-
ples used for the FTIR-DRIFT studies were compres-
sion-moulded with KBr powder.
3.4.4. n-Butyl ferulate. 1H NMR, d, ppm: 0.94 (3H, t,
J = 7.4 Hz,
CH2CH2CH2CH3),
1.40
(2H,
m,
CH2CH2CH2CH3), 1.65 (2H, m, CH2CH2CH2CH3),
3.88 (3H, s, OCH3), 4.18 (2H, t, J = 6.7 Hz,
CH2CH2CH2CH3), 6.23 (1H, s, OH), 6.27 (1H, d,
J = 15.9 Hz, CHCHCOO), 6.88–7.05 (3H, m, aromatic),
7.59 (1H, d, J = 15.9 Hz, CHCHCOO). 13C NMR, d,
ppm: 14.1 (CH2CH2CH2CH3), 19.4 (CH2CH2CH2CH3),
3.4. General procedure for the synthesis of alkyl ferulates
Except for methyl ferulate, which was purchased from
Apin Chemicals Ltd (Abingdon, UK), a solution of
ferulic acid (10.0 g, 0.05 mol) in the appropriate alcohol
(ethanol, n-propanol, iso-propanol, n-butanol, iso-buta-
nol, 2-butanol, 200 mL) in a 500 mL round-bottomed
flask was flushed with nitrogen and immersed in a water-
bath at room temperature. Acetyl chloride (40 mL,
0.5 mol) was added slowly under nitrogen and the reac-
tion stirred for a further 16 h at ambient temperature.
31.0
(CH2CH2CH2CH3)
CHCHCOO), 167.9 (CHCHCOO).
(CH2CH2CH2CH3),
56.2
109.8–148.4
(OCH3),
(aromatic
64.7
and
3.4.5. iso-Butyl ferulate. 1H NMR, d, ppm: 0.97 (6H, d,
J = 6.7 Hz, CH2CH(CH3)2), 2.00 (1H, m,
CH2CH(CH3)2), 3.91 (3H, s, OCH3), 3.98 (2H, d,
J = 6.7 Hz, CH2CH(CH3)2), 6.12 (1H, s, OH), 6.30
(1H, d, J = 15.9 Hz, CHCHCOO), 6.90–7.07 (3H, m,
aromatic), 7.61 (1H, d, J = 15.9 Hz, CHCHCOO). 13C
NMR, d, ppm: 19.5 (CH2CH(CH3)2), 28.1
(CH2CH(CH3)2), 56.3 (OCH3), 71.0 (CH2CH(CH3)2)
Reverse phase TLC of the alkyl ferulates was performed
using RP-18 F254 silica on aluminium backing (Merck),

C. Vafiadi et al. / Tetrahedron: Asymmetry 16 (2005) 373–379
379
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Acknowledgements
We would like to express our gratitude to the General
Secretariat for Research and Technology (GSRT),
Greece, for funding this work.
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