Chemoenzymatic preparation of novel substrates for feruloyl esterases

Article abstract of DOI:10.1055/s-2001-17450

Four novel active esters of 4-hydroxy-3-methoxycin-namic acid as substrates for feruloyl esterases were prepared in a four-step procedure including chemoselective enzymatic deprotection of acetylated compounds by Lipase PS.

Full text of DOI:10.1055/s-2001-17450

LETTER
1559
Chemoenzymatic Preparation of Novel Substrates for Feruloyl Esterases
C
hemoenzymatic Pre
p
á
aration of
N
r
ovel
S
u
i
bstrate
afor Feruloyl
EsteM
rases astihubová,*a Vladimír Mastihuba,b Lubomír Kremnický,a,c Julious L Willet,c Gregory L Côtéc
k
Slovak Academy of Sciences, Institute of Chemistry, Dúbravská cesta 9, 842 38 Bratislava, Slovakia
l
Slovak University of Technology, Faculty of Chemical Technology, Department of Milk, Fats and Food Hygiene, Radlinského 9, 812 37
Bratislava, Slovakia
m
NCAUR, ARS, USDA, 1815 North University Street, Peoria, Illinois 61604, USA
Fax +421(2)59410222; E-mail: chemjama@savba.sk
Received 28 May 2001
conditions (Scheme). No hydrolysis of trifluoroethyl ester
4d was observed,13 but 4-nitrophenyl (4a) and 4-umbel-
liferyl (4b) esters were completely hydrolyzed (Table). 1-
Naphthyl ferulate (4c) was partially cleaved, approxi-
mately 10% of ferulic acid was found in the reaction mix-
ture according to TLC. Subsequent purification of 4c was
complicated by the presence of liberated 1-naphthol.
Abstract: Four novel active esters of 4-hydroxy-3-methoxycin-
namic acid as substrates for feruloyl esterases were prepared in a
four-step procedure including chemoselective enzymatic deprotec-
tion of acetylated compounds by Lipase PS.
Key words: chemoselectivity, esters, enzymatic deprotection, feru-
loyl esterase, ferulic esters
During our work on feruloyl esterases we found that a
commercial preparation of lipase from Burkholderia
cepacia14 (originally classified as Pseudomonas cepacia)
possesses no feruloyl esterase or any other contaminating
hydrolase activity,15 thus allowing it to be used for
deacetylation of 3a–d without risking concurrent hydrol-
ysis of labile ester bonds. Moreover, the enzyme is stable
in chlorinated solvents16 in which the starting esters 3a–d
are soluble. The enzymatic acetyl cleavage by alcoholysis
in organic solvent proceeded almost quantitatively with-
out affecting the other part of molecule. We obtained pure
feruloyl esters 4a–d in high yields (84-91%) after
crystallization17 (Scheme, Table).
Hydroxycinnamic esterases are attracting growing inter-
est as promising biocatalysts in the processing of
hemicelluloses1 and wine2 as well as in the production of
phenolic acids as fine chemicals3,4 from natural resources.
Although not distributed as purified enzymes, they are
present in commercial hemicellulases, pectinases and li-
pases. This fact has generated a demand for new sub-
strates of hydroxycinnamic esterases for use in rapid
assays and enzymatic synthesis. Here we report a facile
and efficient way to prepare 4-nitrophenyl, 4-methylum-
belliferyl, 1-naphthyl and 2,2,2-trifluoroethyl esters of 4-
hydroxy-3-methoxycinnamic acid with chemoselective
enzymatic alcoholysis of the corresponding 4-O-acetylat-
ed ester intermediates as a key deprotection step.
COOH
COCl
COOR
COOR
The synthesis of active esters 4a–d started from ferulic
acid (4-hydroxy-3-methoxy-trans-cinnamic acid, 1). In
the first step, acetylation of 1 was necessary to avoid side
reactions.5 Chloride 2 was then prepared by stirring acety-
lated 1 with SOCl2 in toluene at 75 °C for 2 hours. Effi-
cient esterification (0.85 equiv Et3N–0.25 equiv DMAP in
CH2Cl2)6 gave 3a–d in 88–91% yield after crystallization
from aqueous acetone (Scheme).
i, ii
iii
iv or v
OCH3
+ 1 + ROH
OCH3
OCH3
OCH3
OH
OAc
OAc
OH
1
2
3a-d
4a-d
Scheme Reagents and Conditions: (i) Ac2O, Py, 89%; (ii) SOCl2,
toluene, 75 °C, 2 h, 91%; (iii) ROH, Et3N, DMAP, CH2Cl2, 3 h, 88–
91%; (iv) Pyrrolidine (1.1 equiv), CH2Cl2–EtOH (1:1), r.t., 72 h; (v)
Lipase PS, i-PrOH, CH2Cl2, 40 °C, 72 h, 84–91%
There are numerous mild and selective chemical and en-
zymatic methods described in the literature for deacetyla-
tion of aromatic acetates without affecting aliphatic
esters.7–10 Esters 3a–d, however, represent activated aryl
and alkyl esters sensitive both to acidic and mildly basic
conditions.11 That is why selective deprotection of the hy-
droxycinnamate part of the molecule is a delicate prob-
lem. Treatment of 3a–d (1 mmol) with the 1.1 equivalent
of pyrrolidine12 (added stepwise during three days) in 100
mL ethanol/CH2Cl2 (1:1) showed very low chemoselec-
tivity and high sensitivity of aryl esters 3a–c to mild basic
In conclusion, the simple and efficient chemoselective en-
zymatic deacetylation reaction presented here may be ap-
plicable for alkali labile alkyl or aryl esters of
hydroxycinnamic acid derivatives. The utilization of es-
ters prepared by this route in analytical or synthetic assays
on feruloyl esterases is under investigation.
Acknowledgement
This work was supported in part by the Biotechnology Research and
Development Corporation under Cooperative Research and De-
velopment Agreement number 58-3K95-8-591.
Synlett 2001, No. 10, 28 09 2001. Article Identifier:
1437-2096,E;2001,0,10,1559,1560,ftx,en;G10201ST.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

1560
M. Mastihubová et al.
LETTER
(17) In a typical procedure, Lipase PS (5 g) was added to a
Table Comparison of Chemical and Enzymatic Conditions Used
for Deacetylation of 3a–d
solution of 3a–d (0.003 mol) in CH2Cl2 (50 mL) and 2-
propanol (25 mL). Both solvents were pre-dried over
molecular sieves, 3 Å. The reaction mixture was stirred or
shaken at 250 rpm until complete disappearance of the
starting material, generally for about 3 days at 40 °C. The
reaction was terminated by removal of the enzyme by
filtration. The filter cake was washed with CH2Cl2 (CHCl3
for 4a) and the filtrate concentrated by evaporation of the
solvents at reduced pressure. The crude product was purified
by crystallization from CH2Cl2–cyclohexane and the
enzyme filter cake could be air-dried and reused for at least
five additional reaction cycles.
Substrate
R
Product Pyrrolidine Lipase
Yield (%) Yield (%)b
3a
4a
0a
91
NO2
O
O
3b
4b
0a
84
CH3
The isolation and purification of 4-methylumbelliferyl
ferulate (4b) was more complicated due to its insolubility.
Thus, Lipase PS was washed with DMSO, followed by
addition of water to the filtrate, and the precipitated 4b was
filtered and crystallized from 2-methoxyethanol–CHCl3.
4a: 1H NMR (400 MHz, DMSO-d6): 3.84 (s, 3H, OCH3),
6.75 (d, 1 H, JAB = 15.8 Hz, H-A), 6.84 (d, 1 H, J5,6 = 8.2 Hz,
H-5), 7.23 (dd, 1 H, J2,6 = 2.0 Hz, H-6), 7.44 (d, 1 H, H-2),
7.51 (dt, 2 H, H-2',6'), 7.82 (d, 1 H, H-B), 8.32 (dt, 2 H, H-
3', 5'); 13C NMR (100.6 MHz, DMSO-d6): 55.73 (OCH3),
111.56 (C-2), 112.55 (C-A), 115.57 (C-5), 123.17 (C-2', 6'),
124.00 (C-6), 125.24 (C-3', 5'), 125.29 (C-1), 144.84 (C-4'),
148.01 (C-3), 148.15 (C-B), 150.10 (C-4), 155.62 (C-1'),
164.60 (COO). 4b: 1H NMR (300 MHz, DMSO-d6): 2.46
(s, 3H, CH3), 3.84 (s, 3 H, OCH3), 6.40 (s, 1 H, H-3'), 6.74
(d, 1 H, JAB = 15.9 Hz, H-A), 6.84 (d, 1 H, J5,6 = 8.1 Hz, H-
3c
4c
77b,c
84b
90
89
3d
-CH2-CF3
4d
a No ester was found in the reaction mixture according to TLC, only
products of complete hydrolysis.
b Yield of isolated pure product.
c Ferulic acid and R-OH observed on TLC besides the desired ester.
References and Notes
(1) Faulds, C. B.; Kroon, P. A.; Bartolomè, B.; Williamson, G.
Carbohydrate Biotechnology Protocols, In Methods in
Biotechnology, Vol. 10; Bucke, C., Ed.; Humana Press Inc.:
Totowa NJ, 1998, 183–195.
(2) Okamura, S.; Watanabe, M. Agric. Biol. Chem. 1982, 46,
297.
(3) Kroon, P. A.; Williamson, G. Biotechnol. Appl. Biochem.
1996, 23, 263.
(4) Faulds, C. B.; Bartolomè, B.; Williamson, G. Ind. Crops.
Prod. 1997, 6, 367.
5), 7.23 (dd, 1 H, J2,6 = 1.9 Hz, H-6), 7.26 (dd, 1 H, J5’,6’
=
8.2 Hz, J6’,8’ = 2.0 Hz, H-6’), 7.35 (d, 1 H, H-8’), 7.44 (d, 1
H, H-2), 7.80 (d, 1 H, H-B), 7.84 (d, 1 H, H-5’); 13C NMR
(75.5 MHz, DMSO-d6): 18.13 (CH3), 55.71 (OCH3),
110.08 (C-8’), 111.52 (C-2), 112.80 (C-A), 113.62 (C-3’),
115.56 (C-5), 117.38 (C-10’), 118.45 (C-6’), 123.83 (C-6),
125.32 (C-1), 126.36 (C-5’), 147.78 (C-B), 147.98 (C-3),
149.97 (C-4), 152.99 (C-4’+C-9’), 153.54 (C-7’), 159.66 (C-
2’), 164.84 (COO). 4c: 1H NMR (300 MHz, CDCl3): 3.95
(s, 3 H, OCH3), 5.94 (s, 1 H, OH), 6.65 (d, 1 H, JAB = 15.9
Hz, H-A), 6.97 (d, 1 H, J5,6 = 8.2 Hz, H-5), 7.13 (d, 1 H, J2,6
= 1.9 Hz, H-2), 7.18 (dd, 1 H, H-6), 7.32 (dd, 1 H, J2’,3’ = 7.5
Hz, J2’,4’ = 0.9 Hz, H-2’), 7.46 - 7.53 (m, 3 H, H-6’+H-7’+H-
3’), 7.76 (d, 1 H, J3’, 4’ = 8.3 Hz, H-4’), 7.86 - 7.95 (m, 2 H,
H-5’+H-8’), 7.92 (d, 1 H, H-B); 13C NMR (75.5 MHz,
CDCl3): 55.98 (OCH3), 109.57 (C-2), 114.27 (C-A),
114.85 (C-5), 118.15 (C-2’), 121.35 (C-8’), 123.54 (C-6),
125.45 (C-3’), 125.89 (C-4’), 126.39 (C-6’+C-7’), 126.76 (C-
1), 127.00 (C-9’), 128.01 (C-5’), 134.67 (C-10’), 146.86 (C-
1’+C-3), 147.05 (C-B), 148.44 (C-4), 165.75 (COO). 4d: 1H
NMR (400 MHz, CDCl3): 3.92 (s, 3 H, OCH3), 4.57 (q, 2
H, CH2), 5.95 (s, 1 H, OH), 6.32 (d, 1 H, JAB = 15.9 Hz, H-
A), 6.92 (d, 1 H, J5,6 = 8.2 Hz, H-5), 7.03 (d, 1 H, J2,6 = 2.0
Hz, H-2), 7.09 (dd, 1 H, H-6), 7.70 (d, 1 H, H-B); 13C NMR
(100.6 MHz, CDCl3): 55.94 (OCH3), 60.28 (q, CH2, 2JC-C-
F = 37.0 Hz), 109.42 (C-2), 113.09 (C-A), 114.79 (C-5),
123.15 (q, CF3, 1JC-F = 276.9 Hz), 123.62 (C-6), 126.44 (C-
1), 146.80 (C-3), 146.23 (C-B), 148.51 (C-4), 165.46
(COO).
(5) Hatfield, R. D.; Helm, R. F.; Ralph, J. Anal. Biochem. 1991,
194, 25.
(6) Lu, F.; Ralph, J. J. Agric. Food Chem. 1998, 46, 2911.
(7) Green, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 3rd Ed.; Wiley: New York, 1999, 277–278.
(8) Månsson, P. Tetrahedron Lett. 1982, 23, 1845.
(9) Basavaiah, D.; Bhaskar Raju, S. Synth. Commun. 1994, 24,
467.
(10) Reidel, A.; Waldmann, H. J. Prakt. Chem. 1993, 335, 109.
(11) Especially, 4-nitrophenyl benzoates hydrolyze in mildly
alkaline buffers, see: Cevasco, G.; Guanti, G.; Thea, S.;
Williams, A. J. Chem. Soc., Chem. Commun. 1984, 783.
(12) Milder modification of deacetylated conditions from
literature.6,8
(13) When 10 equivalents of pyrrolidine were added at once, 4d
was partially hydrolyzed too.
(14) Lipase PS from Amano Pharmaceutical (LPSAX04509).
(15) Sugiura, M.; Oikawa, T.; Hirano, K.; Inukai, T. Biochim.
Biophys. Acta 1977, 488, 353.
(16) Secundo, F.; Ottolina, G.; Riva, S.; Carrea, G. Tetrahedron:
Asymmetry 1997, 8, 2167.
Synlett 2001, No. 10, 1559–1560 ISSN 0936-5214 © Thieme Stuttgart · New York