Two efficient ways to 2-O- and 5-O-feruloylated 4-nitrophenyl α-L-arabinofuranosides as substrates for differentiation of feruloyl esterases

Article abstract of DOI:10.1016/S0040-4039(03)00038-8

4-Nitrophenyl 2-O-(E)-feruloyl-α-L-arabinofuranoside 1 and 4-nitrophenyl 5-O-(E)-feruloyl-α-L-arabinofuranoside 2 have been synthesized by two different routes. Monoferuloylation was accomplished by a chemoenzymatic sequence employing a regioselective transesterification catalyzed by lipases. The feruloyl group was introduced to enzymatically prepared 2,3- and 3,5-diacetates of 4-nitrophenyl α-L-arabinofuranoside by reaction with 4-O-acetylferuloyl chloride. Removal of the protecting acetyl groups yielded 1 and 2. An alternative chemical synthesis suitable for preparation of larger quantities of 1 and 2 also is presented. The new substrates represent convenient tools to differentiate feruloyl esterases on the basis of their substrate specificity.

Full text of DOI:10.1016/S0040-4039(03)00038-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1671–1673
Two efficient ways to 2-O- and 5-O-feruloylated 4-nitrophenyl
a-
L
-arabinofuranosides as substrates for differentiation of
feruloyl esterases
Ma´ria Mastihubova´,* Jana Szemesova´ and Peter Biely
Institute of Chemistry, Slovak Academy of Sciences, Du´bravska´ cesta 9, 842 38 Bratislava, Slovakia
Received 21 October 2002; revised 11 December 2002; accepted 20 December 2002
Abstract—4-Nitrophenyl 2-O-(E)-feruloyl-a-
L
-arabinofuranoside 1 and 4-nitrophenyl 5-O-(E)-feruloyl-a-L-arabinofuranoside 2
have been synthesized by two different routes. Monoferuloylation was accomplished by a chemoenzymatic sequence employing a
regioselective transesterification catalyzed by lipases. The feruloyl group was introduced to enzymatically prepared 2,3- and
3,5-diacetates of 4-nitrophenyl a-L-arabinofuranoside by reaction with 4-O-acetylferuloyl chloride. Removal of the protecting
acetyl groups yielded 1 and 2. An alternative chemical synthesis suitable for preparation of larger quantities of 1 and 2 also is
presented. The new substrates represent convenient tools to differentiate feruloyl esterases on the basis of their substrate
specificity. © 2003 Elsevier Science Ltd. All rights reserved.
The hydroxycinnamic acids (HCAs) associated with
plant hemicelluloses play an important role in cell wall
integrity and protection of plant tissues against diges-
tion by plant-invading microorganisms.1a,b It is well
documented that the most abundant HCA is ferulic
acid, (E)-4-hydroxy-3-methoxycinnamic acid (FA). FA
occurs as an ester-linked acid to the C-5 position of
linkages between HCAs and carbohydrates. Esterases
liberating HCAs from plant cell walls have become
common components of hemicellulolytic enzyme sys-
tems in a variety of microorganisms, and feruloyl
esterase (FE) has also been reported to be endogenous
to plants. A few reports indicate that microorganisms
produce at least two types of FEs which differ in the
a-
of a-
b- -Galp residues in pectic substances and galactans,
and to the C-4 position of -Xylp units of xyloglucans.
L
-Araf residues in arabinoxylans, to the C-2 position
affinity for 2-O- and 5-O-feruloylated a-L-Araf
L
-Araf residues in arabinan, to the C-6 position of
residues.2 Substrates for such differentiation of FEs
have so far been isolated from plant cell walls. Their
preparation involves enzymatic steps followed by
tedious purification procedures.
D
D
Nature has designed enzymes that can attack ester
Figure 1.
Keywords: ferulate substrates; feruloyl esterases; lipase transesterification; a-L-arabinofuranosides.
* Corresponding author. Tel.: +4212-5941-0239; fax: +4212-5941-0222; e-mail: chemjama@savba.sk
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(03)00038-8

1672
M. Mastihubo6a´ et al. / Tetrahedron Letters 44 (2003) 1671–1673
In this paper we report a synthesis of 4-nitrophenyl
2-O-(E)-feruloyl-a- -arabinofuranoside 1 and 4-nitro-
phenyl 5-O-(E)-feruloyl-a- -arabinofuranoside 2 (Fig.
1), as an alternative to the isolation of naturally occur-
ing feruloylated oligosaccharides. We assume that the
4-nitrophenyl aglycon moiety in these compounds will
mimic the second carbohydrate residue in a similar
manner to 4-nitrophenyl glycosides, chromogenic sub-
strates of glycosidases. The new FE substrates are
ing in a solution of 10% DMF in 0.1 M phosphate
buffer (pH 6) at 37°C for 12 h (Scheme 1).
L
L
The free hydroxyl groups in diacetates 4 and 7 were
esterified with 4-O-acetylferuloyl chloride9 in DMAP,
Et3N, CH2Cl2 to give 5 and 8 in high yields. A similar
feruloylation was applied in the preparation of 4-nitro-
phenyl ferulate,10 a non-saccharide chromogenic FE
substrate.11 Next, we investigated chemoselective deac-
ylation conditions, which cleaved only acetyl esters and
preserved the ferulate ester bond. After unsuccessful
attempts to deacetylate 5 upon mild treatment with
pyrrolidine,12 a literature survey revealed satisfying
chemoselective and simple transesterification condi-
tions.13 Thus compounds 5 and 8 were deacetylated
with a suspension of potassium carbonate in a 2:1
mixture of dichloromethane/methanol to give the prod-
ucts 1 and 2 in 67 and 76% yields as solids (Scheme 1).
intended to be used in an a-L-arabinofuranosidase-cou-
pled photometric assays.3
The regioselective synthesis of monoferuloylated ara-
binofuranosides 1 and 2 requires the application of
various mild protecting/deprotecting strategies with
groups easily introduced or cleaved. For this purpose,
we chose chemoenzymatic as well as classical chemical
approaches. Commercially available 4-nitrophenyl a-
arabinofuranoside4 (NPh a-
-Araf ) 3 was used as the
starting material in our synthesis.
L-
L
Additionally, we synthesized 1 and 2 from 3, by routes
that are more suitable for large-scale preparations. As
illustrated in Scheme 2, the preparation of 1 started
with protection of the 3 and 5 positions of furanoside 3
by selective simultaneous silylation with 1 equiv. of
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane14a,b in pyr-
idine to give 9 in 85% yield. Introduction of the feruloyl
group to position 2 of 9 was achieved by reaction with
4-O-acetylferuloyl chloride in DMAP, Et3N, CH2Cl2.
Subsequent deacetylation with pyrrolidine in
dichloromethane afforded 10 in high yield. Deprotec-
tive desilylation of 10 with nBu4NF in THF afforded
115 in 78% yield.
In order to shorten the lengthy protection/deprotection
sequences usually associated with saccharide synthesis,
the 2-O- and 5-O-monoferuloylated NPh a- -Araf 1
L
and 2 were first prepared by sequences employing
highly selective enzyme reactions. The use of lipases or
esterases for the regioselective acylation or deacylation
of saccharide hydroxyl groups with comparable reactiv-
ity belongs to the classical tools of carbohydrate chem-
istry.5a–c In addition, enzymes work in neutral, weakly
acidic or weakly basic media (pH range 4–8) and at
room temperature, i.e. under conditions suitable for
compounds sensitive to harsh chemical environments.
Due to the higher sensitivity of nitrophenyl aglycons to
acidic, basic and aqueous conditions, we made use of
the slightly higher reactivity of the primary hydroxy
group towards acylation and coupled the NPh arabino-
furanoside 3 with 4-O-acetylferuloyl chloride directly
under mild reaction conditions.9 After purification of
11 (54%) from the starting compound and by-products
by chromatography on silica gel, the acetyl protecting
group was removed with 10 equiv. of pyrrolidine in
dichloromethane to give monoferuloylated 216 in 90%
yield.
The 3,5-diacetate of NPh a- -Araf 4 which is used as
L
an FA acceptor was obtained by selective acetylation of
the corresponding glycoside 3 catalyzed by Lipase PS-
306 (Amano) in vinyl acetate. The lipase catalyzed
transesterification was carried out for 51 h whilst shak-
ing at 40°C to afford 47 in 75% yield. The 5-monoac-
etate and 2,5-diacetate of
3
were isolated by
chromatography on silica gel in low yields as side
products. The 2,3-diacetate 77 was obtained in 60%
yield by regioselective deacetylation of per-O-acetylated
NPh a-
-Araf 6 using Lipolyve CC8 (Lyven) with shak-
L
Scheme 1. Reagents and conditions: (a) Lipase PS, vinyl acetate, 40°C, 51 h, 75%; (b) 4-O-acetylferuloyl chloride, DMAP, Et3N,
CH2Cl2, rt, 3 h, 90% for 5 and 92% for 8; (c) K2CO3 (2 equiv.), 2:1 CH2Cl2/MeOH, /−5/“10°C, 5 h, 67% for 1 and 76% for 2;
(d) Lipolyve CC, 10% DMF in 0.1 M phosphate buffer pH 6, 37°C, 12 h, 60%.

M. Mastihubo6a´ et al. / Tetrahedron Letters 44 (2003) 1671–1673
1673
Scheme 2. Reagents and conditions: (a) (Cl(i-Pr)2Si)2O, pyridine, rt, 6 h, 85%; (b) 4-O-acetylferuloyl chloride (1.2 equiv.), DMAP
(0.25 equiv.), Et3N (1 equiv.), CH2Cl2, rt, 3 h, 91%, (c) pyrrolidine (10 equiv.), CH2Cl2, rt, 2 h, 86% for 10 and 90% for 2; (d)
n-Bu4F, THF, rt, 1 h, 78%; (e) 4-O-acetylferuloyl chloride, toluene, pyridine, /−5/°C for 3 h than 4°C overnight, 54%.
In conclusion, two methods for the synthesis of 4-nitro-
phenyl 2-O-(E)-feruloyl-a- -arabinofuranoside 1 and 4-
nitrophenyl 5-O-(E)-feruloyl-a- -arabinofuranoside 2
J3,4=5.8 Hz, H-3), 5.77 (s, 1H, H-1), 7.16 (dt, 2H, J=2.2,
L
3.3, and 9.3 Hz, NPh), 8.20 (dt, 2H, J=2.2, 3.3, and 9.3
Hz, NPh).
L
Compound 7: 1H NMR (300 MHz, CDCl3): l 2.15 (s,
are elaborated. The first, a chemoenzymatic route,
seems to be simple and short, however, it requires large
quantitites of enzymes. The second, a chemical route,
which is more efficient and economically more feasible,
is suitable for the preparation of larger quantities of 1
and 2. Compounds 1 and 2 were found to be conve-
nient substrates for determination of activity and differ-
entiation of FeEs according to substrate specificity in a
UV-spectrophotometric assay.3 The coupling of the
3H, COCH3), 2.16 (s, 3H, COCH3), 3.84 (dd, 1H, J4,5a
=
4.1, J5a,5b=12.3 Hz, H-5a), 3.92 (dd, 1H, J4,5b=3.5 Hz,
H-5b), 4.25 (bdd, 1H, J4,5a=4.1, J4,5b=3.5 Hz, H-4), 5.20
(dd, 1H, J2,3=1.8, J3,4=5.1 Hz, H-3), 5.43 (d, 1H, J2,3
=
1.8 Hz, H-2), 5.79 (s, 1H, H-1), 7.15 (dt, 2H, J=2.2, 3.3,
and 9.3 Hz, NPh), 8.21 (dt, 2H, J=2.2, 3.3, and 9.3 Hz,
NPh).
8. Lipase from Candida cylindracea, 2VI1072.
9. Hatfield, R. D.; Helm, R. F.; Ralph, J. Anal. Biochem.
1991, 194, 25–33.
action of FeEs with a- -arabinofuranosidase renders 1
L
and 2 chromogenic substrates of FeEs.3
10. Mastihubova´, M.; Mastihuba, V.; Kremnicky´, L.; Willet,
J. L.; Coˆte´, G. L. Syntlett 2001, 1559–1560.
11. Mastihuba, V.; Kremnicky´, L.; Mastihubova´, M.; Willet,
J. L.; Coˆte´, G. L. Anal. Biochem. 2002, 309, 96–101.
12. Helm, R. F.; Ralph, J.; Hatfield, R. D. Carbohydr. Res.
1992, 229, 183–194.
Acknowledgements
This work was supported by a grant from the Slovak
Grant Agency for Science VEGA 2-7136/20.
13. Condo, A. M.; Baker, D. C.; Moreau, R. A.; Hicks, K.
B. J. Agric. Food. Chem. 2001, 49, 4961–4964.
14. (a) Czernecki, S.; Le Diguarher, T. Synthesis 1991, 683–
686; (b) Kaneko, S.; Kawabata, Y.; Ishii, T.; Gama, Y.;
Kusakabe, I. Carbohydr. Res. 1995, 268, 307–311.
References
1. (a) Fry, S. C. Ann. Rev. Plant Physiol. 1986, 37, 165–186;
(b) Ishii, T. Plant Sci. 1997, 127, 111–127.
2. Williamson, G.; Kroon, P. A.; Faulds, C. B. Microbiol-
ogy 1998, 144, 2011–2023.
3. Biely, P.; Mastihubova´, M.; van Zyl, W. H.; Prior, B. A.
Anal. Biochem. 2002, 311, 68–75.
1
15. Compound 1: H NMR (300 MHz, CDCl3): l 3.82 (dd,
1H, J4,5a=2.9, J5a,5b=12.5 Hz, H-5a), 3.92 (s, 3H,
OCH3), 3.97 (dd, 1H, J4,5b=2.5, J5a,5b=12.5 Hz, H-5b),
4.25–4.33 (m, 2H, H-3, 4), 5.31 (bd, 1H, J2,3=2.0 Hz,
H-2), 5.90 (s, 1H, H-1), 6.30 (d, 1H, JA,B=15.9 Hz, H-A),
6.92 (d, 1H, J5%,6%=8.2 Hz, H-5%), 7.02 (d, 1H, J2%,6%=1.7
Hz, H-2%), 7.07 (dd, 1H, J2%,6%=1.7, J5%,6%=8.2 Hz, H-6%),
7.15 (dt, 2H, J=2.2, 3.3 and 9.3 Hz, NPh), 7.67 (d, 1H,
4. Fielding, A. H.; Hough, L. Carbohydr. Res. 1965, 1,
327–329.
5. (a) Reidel, A.; Waldman, H. J. Prakt. Chem. 1993, 335,
109–127; (b) Hennen, W. J.; Sweers, H. M.; Wang, Y.-F.;
Wong, C.h.-H. J. Org. Chem. 1988, 53, 4939–4945; (c)
Lo´pez, R.; Montero, E.; Sa´nchez, F.; Can˜ada, J.; Ferna´n-
dez-Mayoralas, A. J. Org. Chem. 1994, 59, 7027–7032.
6. Lipase from Burkholderia cepacia (originally classified as
Pseudomonas cepacia), LPSAZ0452412.
J
A,B=15.9 Hz, H-B), 8.20 (dt, 2H, J=2.2, 3.3, and 9.3
Hz, NPh).
1
16. Compound 2: H NMR (300 MHz, CD3OD): l 3.87 (s,
3H, OCH3), 4.06 (dd, 1H, J2,3=3.9, J3,4=6.2 Hz, H-3),
4.23–4.35 (m, 3H, H-2, 4, 5a), 4.44 (dd, 1H, J4,5b=3.2,
J5a,5b=11.7 Hz, H-5b), 5.70 (d, 1H, J1,2=1.5 Hz, H-1),
7. Compound 4: 1H NMR (300 MHz, CDCl3): l 2.12 (s,
3H, COCH3), 2.18 (s, 3H, COCH3), 3.49 (bs, 1H, OH),
4.29 (dd, 1H, J4,5a=4.8, J5a,5b=12.2 Hz, H-5a), 4.41 (dd,
1H, J4,5b=3.0 Hz, J5a,5b=12.2 Hz, H-5b), 4.41–4.46 (m,
1H, H-4), 4.49 (bd, 1H, J2,3=2.5 Hz, H-2), 4.84 (dd, 1H,
6.38 (d, 1H, JA,B=15.9 Hz, H-A), 6.79 (d, 1H, J5%,6%=8.2
Hz, H-5%), 7.05 (dd, 1H, J2%,6%=1.7, J5%,6%=8.2 Hz, H-6%),
7.17 (d, 1H, J2%,6%=1.7 Hz, H-2%), 7.20 (d, 2H, J=9.2 Hz,
NPh), 7.63 (d, 1H, JA,B=15.9 Hz, H-B), 8.20 (d, 2H,
J=9.2 Hz, NPh).