Lipase-catalysed deacetylation of botryodiplodin acetate

Article abstract of DOI:10.1016/j.tetasy.2007.02.008

Enantioselective deacetylation of α-(±)-botryodiplodin acetate was successfully accomplished by means of lipase PS to afford (+)-botryodiplodin and α-(+)-botryodiplodin acetate with high enantiomeric excesses. Enzyme-mediated transesterification of the acetylated molecule with n-butanol, as well as its hydrolysis in several organic solvents, are also reported. The CD spectra of (+)-botryodiplodin and α-(+)-botryodiplodin acetate are also presented.

Full text of DOI:10.1016/j.tetasy.2007.02.008

Tetrahedron: Asymmetry 18 (2007) 447–450
Lipase-catalysed deacetylation of botryodiplodin acetate
Cristina Forzato,a Giada Furlan,a Franco Ghelfi,b Patrizia Nitti,a,* Giuliana Pitacco,a
Fabrizio Roncagliab and Ennio Valentina,*
a
`
Dipartimento di Scienze Chimiche, Universita di Trieste, via Licio Giorgieri 1, I-34127 Trieste, Italy
`
Dipartimento di Chimica, Universita degli Studi di Modena e Reggio Emilia, Via Campi 183, I-41100 Modena, Italy
b
Received 29 January 2007; accepted 14 February 2007
Available online 13 March 2007
Abstract—Enantioselective deacetylation of a-( )-botryodiplodin acetate was successfully accomplished by means of lipase PS to afford
(+)-botryodiplodin and a-(+)-botryodiplodin acetate with high enantiomeric excesses. Enzyme-mediated transesterification of the acet-
ylated molecule with n-butanol, as well as its hydrolysis in several organic solvents, are also reported. The CD spectra of (+)-botryo-
diplodin and a-(+)-botryodiplodin acetate are also presented.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(2R/2S,3R,4S), as it is a 2:3 mixture of a-(2R) and b-(2S)
anomers. Its nonnatural diastereomer epi-botryodiplodin
(ꢀ)-2,6 also present as a 1:2 mixture of a and b anomers,
has the (2R/2S,3S,4S)-configuration. We have recently pro-
posed a new stereoselective synthesis of the acetate 3 of the
a anomer of botryodiplodin in racemic form,8,9 and we
report here on its lipase-catalysed kinetic resolution.
Acylated hemiacetals are known to undergo enzymatic
transformations by lipases at the anomeric position. For
example, the a anomer of 1,2,3,4,6-penta-O-acetyl-D-gluco-
pyranose was selectively hydrolysed at the acetyl group at
C-1 by Pseudomonas fluorescens lipase (PFL),1 while its b
anomer was hydrolysed by Candida antarctica lipase.2 Sim-
ilarly, enantiomerically pure cis- and trans-hemiacetals of
2-bromo-5-hydroxypentanal were obtained by C. antarc-
tica lipase B (CALB)-catalysed kinetic resolution of the
corresponding acetates,3 and acetylated hemiacetals of
substituted cyclopropanone were also kinetically resolved
by lipase.4 On the other hand, enzymatic kinetic resolution
of 6-acetyloxy-2H-pyran-3(6H)-one was achieved by lipase
PS transesterification in hexane/n-butanol.5
2. Results and discussion
Initially, in order to verify the stability of the botryodiplo-
din acetate ( )-3 in water, it was set aside in phosphate
buffer at pH 7.4. After 2.5 h, it was transformed into
botryodiplodin ( )-1 for 40% (HRGC) and after 23 h,
botryodiplodin ( )-1 thus formed was partially (20%)
converted into its diastereomer ( )-2 (Fig. 1). The easy
hydrolysis of b-botryodiplodin acetate in water at room
temperature had already been observed.10
Botryodiplodin (ꢀ)-1 (Fig. 1) is a natural mycotoxin6 with
wide biological activities.7 Its absolute configuration is
O
O
O
HO
botryodiplodin (–)-1
HO
epibotryodiplodin (–)-2
AcO
O
O
O
α-botryodiplodin acetate ( )-3
Figure 1. Botryodiplodin (ꢀ)-1, epi-botryodiplodin (ꢀ)-2 and a-botryodiplodin acetate ( )-3.
*
Corresponding authors. Tel.: +39 040 5583917; fax: +39 040 5583903 (E.V.); tel.: +39 040 5583923; fax: +39 040 5583903 (P.N.); e-mail addresses:
pnitti@units.it; evalentin@units.it
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.02.008

448
C. Forzato et al. / Tetrahedron: Asymmetry 18 (2007) 447–450
In spite of these results, we attempted a lipase PS catalysed
hydrolysis11 of compound 3, to verify whether the enzy-
matic hydrolysis could be faster than the chemical hydroly-
In toluene, the presence of n-butanol had no effect, while in
DME the selectivity was higher without adding n-butanol
(E = 175 vs 86; Table 2, entry 1 and Table 1, entry 1)
but with a longer reaction time, while in the biphasic
system DME/buffer, the reaction was faster but the selec-
tivity was lower. The best conditions were found to be
diisopropyl ether, as the reaction proceeded faster (20 h)
and with high enantioselectivity (E > 200; Table 2,
entry 4).
sis. After 1 h 17 min, the corresponding a-botryodiplodin
25
acetate (+)-3, ½aꢁD ¼ þ22:2 (c 0.18, CHCl3), 33% ee, and
25
botryodiplodin (+)-1, ½aꢁD ¼ þ19:5 (c 0.22, CHCl3), lit.6b
25
25
½aꢁD ¼ ꢀ69 (c 0.85, CHCl3), lit.6c ½aꢁD ¼ ꢀ68 (c 0.35,
MeOH), 18% ee (60% conversion), were recovered. The
enantiomeric excesses were determined by chiral
HRGC.6c,12
In CDCl3 the 1H NMR spectrum6b,c of botryodiplodin (+)-
1 (ratio of the a and b anomers, 2:3; the attributions will be
given later) showed both anomeric protons at 5.18, as a sin-
glet for the b anomer and a doublet of doublets for the a
anomer (0.4H, J1 = 12.1 Hz, J2 = 4.8 Hz) (Fig. 2a). Addi-
tion of a few drops of deuteriated benzene allowed the sep-
aration of the anomeric protons, the dd of the a anomer
being shifted to 5.10 ppm and the singlet of the b anomer
to 4.97 (Fig. 2b). In CDCl3, their respective OH signals res-
onated at 4.87 ppm as a doublet (0.4H, J = 12.1 Hz) and at
2.77 ppm (0.6H) as a broad singlet (Fig. 2a). When benzene
was added, their resonance positions moved to 4.76 and
2.21 ppm, respectively, this latter signal being overlapped
with H-3 of the a anomer (Fig. 2b).
The a-botryodiplodin acetate ( )-3 was then subjected to
lipase PS-catalysed deacetylation reaction with n-butanol5
and MeOH3 in organic solvent. The main results are shown
in Table 1.
The conditions proposed by Parve et al.3 for the enzymatic
deacetylation of hemiacetals of a-bromo-x-hydroxyaldehy-
des with C. antarctica lipase B, namely, MeOH in CHCl3,
gave the lowest selectivity (E = 7), when used with lipase
PS, while the highest activity (E > 200) was observed using
n-butanol as the alcohol and toluene as the organic solvent.
However, although under these conditions the enantio-
meric ratio was high, the reaction rates were low, so we
decided to carry out the hydrolysis of a-( )-3 in both
non-anhydrous organic solvents and in a biphasic system.15
The best results are reported in Table 2.
By the addition of D2O to the sample containing C6D6, the
OH signal of the b-isomer at 2.21 ppm rapidly disappeared,
Table 1. Lipase PS catalysed transesterification of a-botryodiplodin acetate ( )-3
O
O
O
Lipase PS
+
+
n-BuOAc
(MeOAc)
n-butanol
(MeOH)
AcO
HO
AcO
O
O
O
(+)-1
α-(+)-3
α-( )-3
Entry
Solvent
DMEa
Time (d)
Conv.b (%)
eec (%) (+)-1
eec (%) (+)-3
E13
86
>200
35
1
2
3
4
7
7
8
8
47
51
51
17
94
95
85
73
84
99
88
15
Toluenea
a
CH2Cl2
MeOH/CHCl3
3
7
a Conditions: botryodiplodin acetate 3 (0.06 g, 0.32 mmol), 2 mL of organic solvent, n-butanol (0.15 mL, 1.6 mmol), lipase PS (0.05 g).
b Calculated values, Ref. 14.
c Determined by chiral HRGC.
Table 2. Lipase PS catalysed deacetylation of botryodiplodin acetate a-( )-3
Entry
Solvent
Time
Conv.b (%)
eec (+)-1 (%)
eec (+)-3 (%)
E13
1
2
3
4
5
6
DMEa
13 d
15 h
5 d
20 h
4 d
46
52
51
50
53
49
97
90
93d
98
87
97
84
99
97
99
97
93
175
99
115
>200
59
>200
DME/buffer16
Light petroleuma
i-Pr2Oa
THFa
Toluenea
5 d
a Conditions: botryodiplodin acetate 1 (0.06 g, 0.32 mmol), 2 mL of organic solvent, lipase PS (0.05 g). At the end of the reaction lipase PS was filtered and
washed with ethyl acetate.
b Calculated values, Ref. 14.
c Determined by chiral HRGC.
d Botryodiplodin was extracted with ethyl acetate because of its insolubility in light petroleum.

C. Forzato et al. / Tetrahedron: Asymmetry 18 (2007) 447–450
449
4%
H
H
3
Me
4
O
2
O
Me
O
H
Figure 3. a-Anomer of botryodiplodin 1.
The enantiomerically pure a anomer of botryodiplodin
25
acetate 3 showed a positive specific rotation f½aꢁD
¼
þ87:9 (c 1.65, CHCl3)}, while the b anomer of botryo-
diplodin acetate 3 is reported17 to have negative optical
rotation power {[a]D = ꢀ104 (c 0.09, CHCl3)}, thus
suggesting that the sign of the specific rotation does depend
solely on the configuration at C-2. As to the CD spectra
run in MeOH, the a anomer of botryodiplodin acetate
(+)-3 showed two negative Cotton effects at 218 and
283 nm (De218 ꢀ0.2, De283 ꢀ0.4), which can be attributed
to the n!p* transition bands of the acetate group and
the acetyl group, respectively. In the same solvent, botryo-
diplodin (+)-1, which has the carbonyl group as a single
chromophore, showed a positive Cotton effect at 284 nm
(De284 +0.9).
Notwithstanding botryodiplodin acetate 3 is more stable
than botryodiplodin 1, it is sensitive to acidic conditions
and should be manipulated very carefully, and stored pref-
erably in CH2Cl2 or CHCl3 solution at ꢀ20 ꢁC. Interest-
ingly, at 4 ꢁC the pure a anomer of botryodiplodin
acetate 3 slowly converted into the more stable b anomer.
This is rather surprising as acetylated hemiacetals are usu-
ally considered stable structures, at least in sugar chemis-
try. This unusual behaviour could be attributed to the
easy removal of the acetyl group, owing to its leaving
group ability, with generation of an ion pair, which would
eventually lead to the more stable b anomer.
Figure 2. Part of 1H NMR spectrum of botryodiplodin (+)-1: (a) in
CDCl3, (b) in CDCl3/C6D6, (c) the same as (b) with a few drops of D2O
added.
3. Conclusion
whereas that of the a-isomer did not exchange with deute-
rium (Fig. 2c). Strangely enough, it became a singlet and as
a consequence the double doublet at 5.10 ppm became a
doublet. This result was interpreted in terms of the exis-
tence of an intramolecular hydrogen bond involving the
OH group of the a anomer and the carbonyl group at C-
4, which would lower its mobility. The presence of a small
amount of D2O would simply eliminate coupling but would
not be sufficient enough to exchange with OH with the
same velocity as for the b anomer.
In conclusion we have successfully accomplished the enan-
tioselective deacetylation of a-( )-botryodiplodin acetate
by means of lipase PS to afford (+)-botryodiplodin and
a-(+)-botryodiplodin acetate with high enantiomeric
excesses. This method, we have developed, demonstrates
that an easy to hydrolyse acetal function can be resolved
by lipase-catalysed hydrolysis in organic solvent or in a
biphasic system and not only by a transesterification process.
Studies on the enzymatic resolution of botryodiplodin ace-
tate analogues (with alternative alkyl substituents to
methyl at C-3) have been scheduled and will appear in
due course.
Finally, the attribution of the descriptors a and b to the
anomers of botryodiplodin 1 should be noted. This was
made by means of NOE different measurements. Irradia-
tion of the signal relative to H-3 at 2.45 ppm of (0.4H)
caused an enhancement of the H-2 signal (dd, 4%), thus
demonstrating that these two signals belonged to the a
anomer (Fig. 3). Figure 3 also shows the possible orienta-
tion of the OH group which would account for both the
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3
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100 ꢁC for 10 min, 3 ꢁC/min until 150 ꢁC) retention time: (+)-
(2R,3R,4S)-3, 29.01 min and (ꢀ)-(2S,3S,4R)-3, 29.70 min;
(ꢀ)-(2R/S,3R,4S)-1, 25.91 min and (+)-(2R/S,3S,4R)-1,
26.28 min, the two anomers having the same retention time.
13. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am.
Chem. Soc. 1982, 104, 7294–7299.
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C9H14O4 requires C, 58.05; H, 7.58; 1H NMR (CDCl3,
400 MHz): d 1.06 (3H, d, J 7.3 Hz, CH3C(3)), 2.08 (3H, s,
CH3COO), 2.28 (3H, s, CH3C@O), 2.83 (1H, m, J 5.0, 7.2,
9.9 Hz, C(3)H), 3.33 (1H, ddd, J 5.9, 7.5, 9.9 Hz, C(4)H), 4.04
(1H, dd, J 7.5, 9.1 Hz, C(5)H), 4.42 (1H, dd, J 5.9, 9.1 Hz,
C(5)H), 6.22 (1H, d, J 5.0 Hz, C(2)H); 13C NMR (CDCl3,
14. Rakels, J. L. L.; Straathof, A. J. J.; Heijnen, J. J. Enzyme
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15. Faber, K. Biotransformations in Organic Chemistry, 5th ed.;
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16. To a suspension of botryodiplodin acetate 3 (0.300 g,
1.6 mmol) in 10 mL of DME and 5 mL of phosphate buffer
(pH 7.4, 0.1 M), lipase PS (0.250 g) was added under vigorous
stirring. After 15 h, water was added and the reaction mixture
was extracted with ether and ethyl acetate, and the organic
phase dried with anhydrous Na2SO4. After evaporation of the
solvent, the crude mixture was purified by flash chromato-
graphy to afford botryodiplodin acetate 3 (0.08 g, 27%
100 MHz):
d
9.6 (CH3C(3)), 20.6 (CH3COO), 30.0
(CH3C@O), 40.3 (C(3)), 52.4 (C(4)), 68.7 (C(5)), 98.6
(C(2)), 169.8 (COO), 206.7 (C@O); IR (film): 1743 (C@O),
1713 (C@O) cmꢀ1, m/z (EI): 143 (1%, M+ꢀ43), 127 (17), 126
(18), 98 (10), 83 (57), 55 (29), 43 (100).
25
25
yield, ½aꢁ ¼ þ87:9 (c 1.65, CHCl3), ½aꢁD ¼ þ100 (c 0.35,
MeOH), DDe218 = ꢀ0.2 (MeOH), De283 = ꢀ0.4 (MeOH),
25
ee 99%) and botryodiplodin 1 (0.06 g, 26% yield, ½aꢁD ¼ þ65
25
10. Arsenault, G. P.; Althaus, J. R.; Divekar, P. V. Chem.
Commun. 1969, 1414–1415.
(c 0.85, CHCl3), ½aꢁD ¼ þ56:5 (c 0.55, MeOH), De284 = +0.9
(MeOH), ee 90%).
11. To a suspension of botryodiplodin acetate 3 (0.120 g,
0.5 mmol) in 15 mL of phosphate buffer (pH 7.4, 0.1 M),
17. Moreau, S.; Lablanche-Combier, A.; Biguet, J.; Foulon, C.;
Delfosse, M. J. Org. Chem. 1982, 47, 2358–2359.