Catalytic behaviour of chloroperoxidase from Caldariomyces fumago in the oxidation of cyclic conjugated dienes

Article abstract of DOI:10.1016/S0957-4166(02)00509-8

Chloroperoxidase from Caldariomyces fumago has been investigated as a catalyst for the oxidation of cyclic conjugated dienes. The nature of the substituents and the size of the carbocycle affect the enantioselectivity of the enzyme. An unexpected course o

Full text of DOI:10.1016/S0957-4166(02)00509-8

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 1889–1892
Catalytic behaviour of chloroperoxidase from Caldariomyces
fumago in the oxidation of cyclic conjugated dienes
Claudia Sanfilippo* and Giovanni Nicolosi
CNR Istituto Chimica Biomolecolare, Sezione di Catania, Via del Santuario 110, I-95028 Valverde CT, Italy
Received 5 July 2002; accepted 23 August 2002
Abstract—Chloroperoxidase from Caldariomyces fumago has been investigated as a catalyst for the oxidation of cyclic conjugated
dienes. The nature of the substituents and the size of the carbocycle affect the enantioselectivity of the enzyme. An unexpected
course of the CPO-catalyzed oxidation has been observed in the reaction of cis,cis-1,3-cyclooctadiene. © 2002 Published by
Elsevier Science Ltd.
1. Introduction
tiomeric faces and enantiotopic double bonds, was
complete after 48 h and two diols, (−)-1a and (+)-1b
(ratio 1.3:1), were isolated in satisfactory yield and 70%
ee.7 The chiral monoepoxide was not detected, possibly
due to its rapid hydrolysis under the reaction condi-
tions (Scheme 1).
Chloroperoxidase (CPO) from Caldariomyces fumago is
a promising biocatalyst for asymmetric oxidation.1,2
The epoxidation of olefins with hydroperoxides is the
most studied reaction catalyzed by CPO, since chiral
epoxides are important intermediates in asymmetric
syntheses of biologically or pharmaceutically active
compounds.
Taking this result into consideration, we decided to
investigate the behavior of different conjugated cyclic
dienes. We first considered diol 2 (Table 1), in which
the presence of the two cis-substituents reduces the
molecular symmetry but retains the enantiotopic nature
of the double bonds. The CPO-catalyzed oxidation of 2
proceeded with a lower reaction rate with respect to
that of 1. The expected epoxide 2a was detected in
traces, and after 72 h a single product was isolated in
25% yield.8 NMR analysis and the specific rotation
value allowed us to define this product as 5-cyclohexen-
1a,2b,3a,4b-tetrol, (+)-2b, indicating CPO-attack only
to the double bond anti to the original hydroxyl groups.
The 78% ee determined for (+)-2b revealed good enan-
tiotopic CPO-recognition regarding the two double
bonds of the substrate.
The CPO-catalyzed oxygen transfer to carbonꢀcarbon
double bonds has been investigated on a broad range of
alicyclic mono-olefins, and the effects of chain length,
double bond position and cis/trans-stereochemistry on
the enantioselectivity and reactivity of the enzyme were
observed.3–6
In our previous work we described the CPO-catalyzed
epoxidation to 1,3-cyclohexadiene, the first example of
a biocatalytic oxidation of a cyclic conjugated diene.7
The satisfactory results we obtained in terms of the
chemical yields and ee of the products prompted us to
extend the methodology to substituted cyclohexadienes
and larger cyclic conjugated systems.
2. Results and discussion
2.1. The role of substituents in the oxidation of 1,3-
cyclohexadiene
CPO-catalyzed oxidation with tert-butyl hydroperoxide
(t-BHP) of 1,3-cyclohexadiene 1, having pro-enan-
Scheme 1. Oxidation of 1,3-cyclohexadiene catalyzed by
CPO.
* Corresponding author. E-mail: sanfilippo@issn.ct.cnr.it
0957-4166/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0957-4166(02)00509-8

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C. Sanfilippo, G. Nicolosi / Tetrahedron: Asymmetry 13 (2002) 1889–1892
Table 1. Oxidation of 1,3-cyclohexadienes catalyzed by CPOa
In a subsequent experiment, the diacetate 3 was tested
as a substrate. After 48 h, complete conversion was
observed with formation of two reaction products, 3a
and 3b. In this case the intermediate epoxide, 3a (10%),
was evidenced in solution and its anti configuration
determined by comparison of the spectroscopic proper-
ties with those of an authentic sample (prepared by
oxidation of 3 with m-CPBA).9 Formation of a single
epoxide again demonstrated the high facial-selectivity
in the oxygen transfer from enzyme to substrate. Chiral
GC and NMR analyses evidenced that both 3a and 3b
were racemic mixtures, thus indicating the detrimental
effect of the acetyl groups on the enantiotopic recogni-
tion of the two double bonds of 3 by the enzyme. With
the aim of better evaluating this effect, we considered
the methoxy derivative 4 as a substrate. The oxidation
reaction gave low conversion (9%) with formation of a
single epoxide, 4a. Its structure was assigned by com-
parison with a synthetic sample obtained by epoxida-
tion of 4 with m-CPBA. In this case the chiral GC
analysis indicated that a racemic mixture had also
formed. No hydrolysis product was detected in the
reaction mixture.
2.2. Enzymatic oxidation of larger cycles
The study of the CPO-catalyzed oxidation was then
extended to larger cyclic dienes, namely 1,3-cyclohepta-
diene 5 and cis,cis-1,3-cyclooctadiene 6.
For cycloheptadiene 5 a 60% conversion of substrate
was observed after 72 h. Cycloheptene oxide was not
detected in the reaction mixture and only diols (−)-5a,
5b and (+)-5c in a ratio of 2:1:1 were observed (Scheme
2).
As in the case of cyclohexadiene, it is possible to
presume the formation of a parent epoxide, which
undergoes rapid chemical hydrolysis with partial rear-
rangement to give the 1,2- and 1,4-diols. The same 79%
ee value measured for (−)-5a and (+)-5c indicates that a
regiospecific hydrolytic oxirane ring opening occurs.
Scheme 2. Oxidation of 1,3-cycloheptadiene catalyzed by CPO.

C. Sanfilippo, G. Nicolosi / Tetrahedron: Asymmetry 13 (2002) 1889–1892
1891
The course of the CPO-catalyzed oxidation of cis,cis-
1,3-cyclooctadiene 6 is quite surprising, since after 6
days a 53% conversion of the substrate was observed
with formation of a product different from the expected
epoxide or diols in 43% yield (Scheme 3), together with
small amounts (5%) of the racemic alcohol 6b. The
main product was identified as 6a by comparison with
a synthetic standard, while the absence of any optical
rotatory power highlighted its racemic nature.10,11
evidenced for the acetoxy- and methoxy derivatives 3
and 4.
With the larger ring system cis,cis-1,3-cyclooctadiene,
6, the biocatalyzed reaction with C. fumago chloroper-
oxidase followed an unexpected course, permitting per-
oxidation at the allylic position. This aspect of the
behavior of CPO is presently under investigation in our
laboratory.
4. Experimental
4.1. Material and methods
Scheme 3. Oxidation of cis,cis-1,3-cyclooctadiene in the pres-
ence of CPO.
CPO from C. fumago (suspension in 0.1 M sodium
phosphate buffer, pH 4) was purchased from Sigma.
Substrates 2, 5 and 6 were products of Aldrich. Ester 3
was obtained by conventional acetylation of cis-1,3-
dihydroxycyclohexadiene 2 with pyridine and acetic
anidride; methoxy derivative 4 was prepared by methy-
lation of 2 with methyl iodide in the presence of a
catalytic amount of cesium carbonate. t-Butyl
hydroperoxide was an Aldrich solution 70% v/v in
water.
The unexpected course of the reaction prompted us to
initiate a deeper investigation. When the reaction was
performed in the absence of CPO, only a 7% yield of 6a
was obtained after 6 days, thus confirming the contri-
bution of CPO to the product formation. In a subse-
quent experiment we treated 6b with t-BHP in the
presence of CPO and no trace of 6a could be detected.
Finally, two separate experiments were performed in
the presence of t-BuOH or TROLOX® as radical scav-
engers.12 Both reactions, as reported in Table 2, gave
amounts of 6a comparable with the value observed
under the standard conditions. The above consider-
ations suggest that a direct, but non-selective, transfer
of -OOH from CPO to the substrate outside the active
site occurs, similar to that observed in CPO-catalyzed
oxidative halogenation reactions.12
Chiral GC and GC–MS analyses were performed on
Megadex DMP b-dimethylpenthyl bCDX OV1701 cap-
illary column and ZEBRON™ 5%-phenyl–95%-
dimethylpolisiloxane, respectively. 1H and 13C NMR
spectra were recorded in CDCl3 or CD3OD at 250.13
and 62.9 MHz, respectively, on a Bruker AMX-250
instrument using TMS as internal reference. Chiral
NMR shift reagent was europium(III) tris-[3-(hepta-
fluoropropyl)hydroxymethylene]-(+)-camphorate, Eu-
(hfc)3. Optical rotations were measured on a DIP 135
JASCO instrument.
Table 2. Oxidationa of 6 with CPO/t-BHP in the presence
of radical scavengers
Radical scavenger
Time (h)
% 6ab
4.2. Enzymatic oxidation of cyclic dienes
None
72
72
72
31
27
34
20% t-BuOH
In a typical oxidation reaction diene (0.1 mmol) was
added to citrate buffer solution (0.1 M, pH 5.0, 2 ml)
under magnetic stirring at room temperature. After a
few minutes CPO (400 U) was added and then t-BHP
(0.1 mmol in two aliquots at 24 h intervals). The
conversion of the substrates was monitored by GC
analysis of aliquots extracted by diethyl ether and using
n-decane as internal standard. Reactions were stirred
vigorously at room temperature (25°C) until profiles of
the reactions were unchanged according to GC
analysis.
TROLOX® 7.5 mM
a 0.2 mmol of 6; CPO 800 U, citrate buffer 0.1 M pH 5; t-BHP, 0.2
mmol, was added to the reaction mixture in two aliquots at 24 h
intervals.
b The reaction conversions were determined from the 1H NMR
spectra of aliquots of the reaction mixture extracted directly with
CDCl3.
3. Conclusions
Preparative scale experiments (2 mmol) were performed
without the use of internal standards. The reactions
were stopped at a defined time, extracted with diethyl
ether or ethyl acetate, the organic solutions dried over
Na2SO4, evaporated to dryness, and the residue sub-
jected to silica gel chromatography eluting with ethyl
acetate/hexane in different ratios.
The results obtained reveal that the activity of Caldari-
omyces fumago peroxidase in the enantioselective oxida-
tion of meso-1,3-cyclodienes is strongly influenced by
the nature of the substrate. Selective epoxidation of one
double bond of the diene is the main activity of the
enzyme, with good enantioselectivity for C6 and C7
1,3-cyclic dienes 1 and 5. The presence of substituents
on the ring affects the stereoselectivity of the reaction,
allowing enantioselectivity in the case of dihydroxy
derivative 2, whereas no selective desymmetrization was
4.2.1. (1R,2S,3S,4S)-Tetrahydroxycyclohex-5-ene, (+)-
2b. Obtained in 25% yield and 78% ee by a 96 h
1
reaction. [h]D=+55.7 (c 0.2 in MeOH). H, 13C NMR

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C. Sanfilippo, G. Nicolosi / Tetrahedron: Asymmetry 13 (2002) 1889–1892
and optical rotation data were in agreement with the
determined by chiral GC analysis of the corresponding
alcohol obtained by reduction with triphenylphosphine
in diethyl ether.17
literature values for (+)-Conduritol F.13
4.2.2. (1a,2a,3b,4a)-1,2-Diacetoxy-3,4-dihydroxycyclo-
hex-5-ene, 3b. Compound 3b was prepared by a 48 h
reaction and was isolated in 65% yield, racemic (by GC
1
Acknowledgements
chiral analysis). H NMR: l 2.03 (s, 3H), 2.08 (s, 3H)
3.97 (dd, J=11.0, 7.5 Hz, 1H), 4.21 (dt, J=7.5, 2.0 Hz,
1H), 4.90 (dd, J=11.0, 4.5 Hz, 1H), 5.58 (dd, J=5.0,
4.5 Hz, 1H), 5.77 (ddd, J=10.0, 5.0, 2.0 Hz, 1H), 5.94
(dd, J=10.0, 2.0 Hz, 1H). Anal. calcd for C10H14O6: C,
52.17; H, 6.13. Found: C, 52.08; H, 5.90%.
This work has been co-funded by MURST (Roma)
within the Project ‘Materiali Innovativi-Metodologie e
Diagnostiche per Materiali ed Ambiente’.
4.3. (1R,2R)-Cyclohept-3-ene-1,2-diol, (−)-5a, (1R,4S)-
cyclohept-2-ene-1,4-diol 5b, (1R,4R)-cyclohept-2-ene-1,4-
diol, (+)-5c
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