Enantioselectivity and diastereoselectivity in the transesterification of secondary alcohols mediated by feruloyl esterase from Humicola insolens

Article abstract of DOI:10.1016/j.tetlet.2004.02.034

Examination of the α and β selectivity of Ferrulic acid esterase (FAE) towards secondary alcohols which contain two stereocenters, at the α and β carbons, reveals that the factor governing the order in the R configuration of the stereogenic center which b

Full text of DOI:10.1016/j.tetlet.2004.02.034

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2755–2757
Enantioselectivity and diastereoselectivity in the transesterification
of secondary alcohols mediated by feruloyl esterase from
Humicola insolens
Nikos S. Hatzakis and Ioulia Smonou*
Department of Chemistry, University of Crete, Iraklion 71409 Crete, Greece
Received 21 January 2004; accepted 9February 2004
Abstract—Examination of the a and b selectivity of Ferrulic acid esterase (FAE) towards secondary alcohols which contain two
stereocenters, at the a and b carbons, reveals that the factor governing the order is the R configuration of the stereogenic center
which bears the alcoholic group.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Ferulic acid esterases1 (FAE), whose physiological role
is to hydrolyze ester linkage between hydroxycinnamic
acids and sugars,2 are a subclass of the carboxylic acid
esterase (EC 3.1.1.1). Their active site and reaction
mechanism are thought to be identical to those of car-
boxylic acid esterases, as deduced by crystal structure
analysis.3;4 These studies have shown that feruloyl
esterases hydrolyze the esters via an acyl–enzyme inter-
mediate, which is a well-known mechanism for serine
hydrolases.5 Despite their similarities with lipases,
ferulic acid esterases do not show lipase activity6 and
exhibit high specificity for hydroxycinnamic acid esters.7
Numerous studies9 have shown that the resolution of
secondary alcohols catalyzed by lipases and esterases is
based on the relative size of the substituents at the ste-
reocenter (Fig. 1a), and their stereopreference can be
predicted10 by the ÔKazlauskas modelÕ, which has been
validated experimentally. Although this model predicts
accurately the stereoselectivity of a lipase towards
alcohols containing one stereocenter (a-carbon), it does
not include alcohols with two neighboring stereocenters
(a and b). Elaboration of this rule to include the b-ste-
reocenter has been done recently11 (Figure 1b).
In our previous publication12 we reported the effect of
the b-stereocenter in the hydrolysis by Candida rugosa
lipase (CRL) of acetates of secondary alcohols, both the
enantio- and the diastereoselectivity. Our results were
incorporated by Schmid in the proposal of his model.
Recently,8 we showed that feruloyl esterase from
Humicola insolens catalyzes with good to excellent
enantioselectivity the transesterification of secondary
alcohols, which are substrates that bear no structural
similarity to the natural substrates of this enzyme. The
observed enantiopreference of FAE was R.
In this work we have examined the a and b selectivity of
Ferrulic acid esterase (FAE) towards secondary alco-
hols, which contain two stereocenters, at the a and b
carbons. For this purpose we synthesized a 50:50 threo/
erythro mixture of 3-phenyl-2-butanol (1) and a 65:35
threo/erythro mixture of 3-m-tolyl-2-butanol (2) and
studied their enzymatic transesterifications with the
activated acyl donor vinyl acetate (Fig. 2). Enantio- and
diastereoselectivities of the transesterifications of the
alcohols 1 and 2 were determined by gas chromato-
graphic analysis of the reaction mixture by using a
permethylated b-cyclodextrin 30 · 0.25 mm, chiral col-
umn. In the reaction of substrate 2 decaline was used
as the internal standard for the calculation of the
H
OH
L
H
OH
Me
M
Me
Ar
1a
1b
Figure 1.
Keywords: Feruloyl esterase; Enantioselectivity; Diastereoselectivity;
Secondary alcohols.
* Corresponding author. Tel.: +30-810-393610; fax: +30-810-393601;
e-mail: smonou@chemistry.uoc.gr
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.02.034

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N. S. Hatzakis, I. Smonou / Tetrahedron Letters 45 (2004) 2755–2757
3
FAE
2
+
+
+
10%conv.
OAc
OH
OAc
OAc
OAc
OAc
1
(2R,3R)-3
(2R,3S)-3
(2S,3S)-3
(2S,3R)-3
80.2%
11.9%
7.9%
<0.1%
β-selectivity
α-selectivity
FAE
8%conv.
OAc
3
2
+
+
+
OH
OAc
OAc
OAc
OAc
2
(2R,3S)-4
16.9%
(2R,3R)-4
74.4%
(2S,3S)-4
(2S,3R)-4
<0.1%
8.7%
β-selectivity
α-selectivity
Figure 2.
Table 1. Asymmetric enzymatic transesterification of substrates 1 and 2 with FAE
Substrate
Time (days)
Conversion (%)
a-Selectivity (% ee)
b-selectivity (% ee)
Diastereoselectivity (% de)
76
1
2
3
3 5
10
>95
74
9>9
66
66
ꢀ
ꢁ
enantio-selectivity and diastereoselectivity. The results
of the enzymatic reactions are summarized in Table 1
and the chromatographic data in Figure 3.
½RRŠ À ½RSŠ
100% :
½RRŠ þ ½RSŠ
The diastereoselectivities of 76% de and 66% de––with
erythro stereochemistry––for substrates
respectively, were calculated as follows:
1
and 2,
The a-selectivity was determined from the relative
amounts of the produced isomers (2R,3R)-3, (2S,3R)-3
and (2R,3R)-4, (2S,3R)-4, coming from the reactions of
substrates 1 and 2, respectively. For example, the >95%
value from substrate 1 was calculated as follows:
ꢀ
ꢁ
ð½RRŠ þ ½SSŠÞ À ð½RSŠ þ ½SRŠÞ
100% :
ð½RRŠ þ ½SSŠÞ þ ð½RSŠ þ ½SRŠÞ
The observed R stereoselectivity8 of the a-stereocenter,
for each individual diastereoisomer, is in accord with the
proposed model by Kazlauskas10 for the faster reacting
enantiomer in the lipase catalyzed transesterification of
secondary alcohols. As shown in Figure 1, for substrate
1, the (R,R) stereoisomer reacts the fastest, followed by
(R,S) and (S,S), which show small differences in reac-
tivity, and finally by the (S,R) stereoisomer. Substrate 2
shows the same reactivity pattern with a small decrease
ꢀ
ꢁ
½RRŠ À ½SRŠ
½RRŠ þ ½SRŠ
100% :
Analogously was calculated and the >95% from sub-
strate 2. The b-selectivity was calculated from the rela-
tive amounts of the (2R,3R)-3 (2R,3S)-3 and (2R,3R)-4
(2R,3S)-4 isomers. For example, the 74% ee was calcu-
lated as follows:
(R,R)-3
-3
(S,S)-3
(R,S)-3
A
B
56
58
Time (min.)
60
62
54
56
58
Time (min.)
60
62
Figure 3. (A) GC analysis of the four diastereomeric acetates 3. (B) Determination of the enantioselectivity and diastereoselectivity of the enzymatic
transesterification using a permethylated b-cyclodextrin 30 · 0.25 mm, chiral column. The retention time of each individual stereoisomer was assigned
by comparison with the corresponding esters 3 and 4 coming from the enzymatic reaction with CRL.12

N. S. Hatzakis, I. Smonou / Tetrahedron Letters 45 (2004) 2755–2757
2757
Van Den Hombergh, J. P. T. W.; Viser, J. Appl. Envirom.
Microbiol. 1997, 63, 4683; (b) Williamson, G.; Faulds, C.
B.; Kroon, P. A. Biochem. Soc. Trans. 1998, 26, 205; (c)
Kroon, P. A.; Williamson, G.; Fish, N. M.; Archer, D. B.;
Belshaw, N. J. Eur. J. Biochem. 2000, 267, 6741; (d) Yu,
P.; Maenz, D. M.; McKinnon, J. J.; Racz, V. J.;
Christensen, D. A. J. Agric. FoodChem. 2002, 50, 1625.
3. Prates, J. A. M.; Tarbouriech, N.; Charnock, S. J.; Fontes,
C. M. G. A.; Ferreira, L. M. A.; Davies, G. J. Structure
2001, 9, 1183.
in selectivity. Analogous studies performed with Pseu-
domonas fluorescence lipase (PFL)13 for substrate 1
revealed that the reactivity pattern was (R,S) > (R,R) >
(S,S) > (S,R), that is the (R,S) isomer reacts faster than
the (R,R) isomer rather than slower. Our recent studies12
with CRL for substrates 1 and 2 proved the reactivity
order to be (R,R) > (S,S) > (R,S) > (S,R), a pattern that
is similar to that of FAE, not PFL.
4. Schubot, F. D.; Kataeva, I. A.; Blum, D. L.; Shah, A. K.;
Ljungdahl, L. G.; Rose, J. P.; Wang, B.-C. Biochemistry
2001, 40, 12524.
5. Cygler, M.; Grochulski, P.; Kazlauskas, R. J.; Schrag, J.
D.; Bouthillier, F.; Rubin, B.; Serreqi, A. N.; Gupta, A. K.
J. Am. Chem. Soc. 1994, 116, 3180.
6. Aliwan, F.; Kroon, P. A.; Faulds, C. B.; Pickersgill, R.;
Williamson, G. J. Sci. FoodAgric. 1999, 79, 457.
7. Kroon, P. A.; Faulds, C. B.; Brezillon, C.; Williamson, G.
Eur. J. Biochem. 1997, 248, 245.
In conclusion, our results clearly demonstrate that the
factor governing the order of reactivity in FAE cata-
lyzed transesterifications of secondary alcohols is the R
configuration of the stereogenic center, which bears the
alcoholic group. The observed diastereoselectivity as
well as the reactivity pattern can be accurately predicted
by the model proposed by Pleiss and collaborators.11
8. Hatzakis, N. S.; Dafnomili, D.; Smonou, I. J. Mol. Cat. B:
Enzymatic 2003, 21, 309.
Acknowledgements
9. (a) Burges, K.; Jennings, L. D. J. Am. Chem. Soc. 1991,
113, 6129; (b) Nishizawa, K.; Ohgami, Y.; Matsuo, N.;
Kisida, H.; Hirohara, H. J. Chem. Soc., Perkin Trans. 2
1997, 1293; (c) Lundell, K.; Raijola, T.; Kanerva, L. T.
Enzyme Microb. Technol. 1998, 22, 86.
We thank the Greek Secretariat of Research and Tech-
nology (PENED 2001) and the Ministry of Education
(B-EPEAEK graduate program) for financial support
and graduate fellowship to N.S.H.
10. Kazlauskas, R. J.; Weiissfloch, A. N. E.; Rappaport,
A. T.; Cuccia, L. A. J. Org. Chem. 1991, 56, 2656.
11. Schulz, T.; Schmid, R. D.; Pleiss, J. J. Mol. Model 2001, 7,
265.
References and notes
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Biotechnol. 1987, 26, 158.
12. Angelis, Y. S.; Smonou, I. Tetrahedron Lett. 1998, 39,
2823.
2. (a) Vries, R. P.; Michelsen, B.; Poulsen, C. H.; Kroon,
P. A.; Heuvel, R. H. H.; Faulds, C. B.; Williamson, G.;
13. Weissfloch, A. N. E.; Kazlauskas, R. J. J. Org. Chem.
1995, 60, 6959.