A fluorescence-based assay for Baeyer-Villiger monooxygenases, hydroxylases and lactonases

Article abstract of DOI:10.1002/adsc.200505040

Alkylation of umbelliferone and nitrophenol with chloroacetone, 3-chlorobutanone, 2-chlorocyclopentanone and 2-chlorocyclohexanone gave the corresponding 2-coumaryloxy and 2-nitrophenoxy ketones. The 2-coumaryloxy ketones were used as fluorogenic substrates to detect Baeyer-Villiger monooxygenases activities of microbial cultures in highthroughput using microtiter plates. The 2-coumaryloxy ketones were oxidized by microorganisms producing Baeyer-Villiger monooxygenases (BVMO), releasing umbelliferone as a fluorescent signal. The substrates were also biotransformed by a microbial monooxygenase (Trichosporon cutaneum). Chemical Baeyer-Villiger oxidation of 2-coumaryloxy ketones using meta-chloroperbenzoic acid proceeded regioselectively to the corresponding acyloxyalkyl derivatives of umbelliferone and nitrophenol. These chiral lactones underwent a fluorogenic and chromogenic reaction upon hydrolysis by esterases, in particular pig liver esterase. Enantioselectivity of the ester hydrolysis reaction was determined by chiral-phase analysis of the unreacted lactones.

Full text of DOI:10.1002/adsc.200505040

FULL PAPERS
A Fluorescence-Based Assay for Baeyer–Villiger
Monooxygenases, Hydroxylases and Lactonases
Renaud Sicard,a Lu S. Chen,b Anita J. Marsaioli,b,* Jean-Louis Reymonda,*
a
Department of Chemistry & Biochemistry, University of Berne, Freistrasse 3, 3012 Berne, Switzerland
Fax: (þ41)-31-631-80-57, e-mail: reymond@ioc.unibe.ch
b
Chemistry Institute, State University of Campinas, CP 6154, 13083-970 Campinas – SP, Brazil
Received: January 21, 2005; Accepted: March 30, 2005
Supporting Information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: Alkylation of umbelliferone and nitrophe- Baeyer–Villiger oxidation of 2-coumaryloxy ketones
nol with chloroacetone, 3-chlorobutanone, 2-chloro- using meta-chloroperbenzoic acid proceeded regiose-
cyclopentanone and 2-chlorocyclohexanone gave the lectively to the corresponding acyloxyalkyl deriva-
corresponding 2-coumaryloxy and 2-nitrophenoxy ke- tives of umbelliferone and nitrophenol. These chiral
tones. The 2-coumaryloxy ketones were used as fluo- lactones underwent a fluorogenic and chromogenic
rogenic substrates to detect Baeyer–Villiger mono- reaction upon hydrolysis by esterases, in particular
oxygenases activities of microbial cultures in high- pig liver esterase. Enantioselectivity of the ester hy-
throughput using microtiter plates. The 2-coumary- drolysis reaction was determined by chiral-phase
loxy ketones were oxidized by microorganisms pro- analysis of the unreacted lactones.
ducing Baeyer–Villiger monooxygenases (BVMO),
releasing umbelliferone as a fluorescent signal. The
substrates were also biotransformed by a microbial Keywords: biotransformations; enzyme catalysis; fluo-
monooxygenase (Trichosporon cutaneum). Chemical rescent probes; lactones; oxidation
oxopentyl ether and an auxiliary enzyme, an alcohol de-
Introduction
hydrogenase.
In all of these assays a sequence of secondary reaction
Most enzymes of industrial interest are currently isolat- steps converts the primary product of the enzyme-cata-
ed from microbial sources or from genetic libraries.[1] In lyzed reaction to a fluorescent product. While this fea-
the search for novel enzymes, it is necessary to apply ture provides highly selective assays for reactions that
functional tests for high-throughput screening that are often cannot be rendered fluorogenic otherwise, it also
capable of detecting enzyme activities with high selec- complicates the reaction set-up and the kinetic analysis.
tivity and sensitivity.[2] Recently we developed enzyme Recently we reported that acyloxymethyl ethers of um-
substrates that release either umbelliferone as a blue- belliferone such as 3, which we originally used to discov-
fluorescent product or nitrophenol as a yellow colored er catalytic antibodies by screening cell culture superna-
product by b-elimination from an intermediate carbonyl tants,[14] are useful fluorogenic probes for lipases and es-
product formed after the enzyme-catalyzed step. This terases (Scheme 1).[15] In this case the primary hydroxy-
assay methodology detects various enzyme types with methyl ether product is very unstable and immediately
high selectivity, including alcohol dehydrogenases,[3] al- releases umbelliferone 4 without any kinetic delay or
dolase catalytic antibodies,[4] acylases and phosphatas- auxiliary reagent.
es,[5] proteases,[6] lipases and esterases,[3,5,7] epoxide hy-
Herein we report the preparation and evaluation of
drolases,[5,8] and transaldolases.[9] These assays may cyclic and acyclic 2-coumaryloxy-ketones 1 and 5–7 as
also be applied in parallel using arrays of structurally di- fluorogenic substrates for detecting Baeyer–Villiger
verse substrates to produce enzyme-specific activity fin- monooxygenase activities using the same principle
gerprints.[10,11] The same fluorescence release principle (Scheme 1). The Baeyer–Villiger oxidation, the oxida-
has also been used by other laboratories in fluorescence tive transformation of a ketone to the corresponding es-
assays for transketolases[12] and Baeyer–Villiger mono- ter or lactone, is a useful transformation in organic syn-
oxygenases.[13] In the latter case Baeyer–Villiger mono- thesis. An asymmetric version of this reaction is possible
oxygenase activity was detected with umbelliferone 4- applying biocatalytic methods with BVMO allowing the
Adv. Synth. Catal. 2005, 347, 1041–1050
DOI: 10.1002/adsc.200505040
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Renaud Sicard et al.
FULL PAPERS
on our previous experiments with such esters,[15] this
product would be relatively unstable under whole-cell
conditions and would readily hydrolyze to form umbel-
liferone 4 as a fluorescent product. This scheme should
be applicable for a variety of 2-coumaryloxy ketones
featuring aliphatic substituents around the reacting car-
bonyl groups, which are usually the reactive substrate
class for BVMO.
Substrate Synthesis
The synthesis of the target ketones was accomplished in
one step by alkylation of 2-chloro ketones 15–18 with ei-
ther umbelliferone or p-nitrophenol as sodium salts in
DMF (Scheme 2). BV oxidation of the ketones with
m-CPBA proceeded regioselectively to afford the corre-
sponding ester and lactones as reference products. An
attempted synthesis of 1,3-bis-umbelliferyloxy-2-propa-
none from 1,3-dichloro-2-propanone did not give any
product. Considering that the lactones were worthy of
investigation as chiral probes for esterases and lipases,
the nitrophenol-derived lactones 13 and 14 were also
prepared by the same synthetic route.
BV-Detection in Microbial Culture
Scheme 1. Substrates and principle of BVMO and esterase
fluorescence assay.
The BVenzymatic activity was investigated in a series of
15 microbial strains, some of which were known to con-
preparation of chiral lactones from achiral ketones.[16,17]
Up to now only two methods have been reported to as-
say BVMO in high-throughput,[13,18] which is a prerequi-
site for microbial screening or directed evolution efforts
in this area. The use of our fluorogenic BV-substrates,
e.g., 1, is demonstrated by the direct detection of BV-ac-
tivity in microbial cell cultures. In addition, the inter-
mediate lactones, e.g., 2, provide chiral fluorogenic
and chromogenic probes for esterases.
Results and Discussion
BV-Assay Design
BVMO are mostly membrane-bound enzymes that re-
quire a cofactor and a cofactor regeneration system.
As a consequence these enzymes are assayed and used
in whole cell system. A Baeyer–Villiger oxidation reac-
tion would therefore become fluorogenic if the primary
oxidation product could release a fluorescent product
under whole-cell reaction conditions. We envisioned
that 2-coumaryloxy ketones such as 1 should be prefer-
entially oxidized to the acyloxymethyl ether of umbelli-
ferone 2 due to the stronger group migration ability of
Scheme 2. Synthesis of BVMO and esterase substrates. Con-
ditions: a) umbelliferone or p-nitrophenol, NaI, K2CO3, ace-
tone, 258C, 5 h (70–97%); b) m-CPBA, NaHCO3, CH2Cl2,
the secondary ether carbon center (Scheme 1). Based 08C to 258C, 15 h (70–87%).
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Fluorescence-Based Assay for Baeyer–Villiger Monooxygenases
tain a monooxygenase activity.[19] The pure cultures
FULL PAPERS
Table 1. Fluorescence microtiter-plate assay in whole cells
using 2-coumaryloxy ketones 1 and 5–7 as substrates.[a]
˜
were acquired from the CCT collection (CCT – ColeÅao
˜
de Culturas Tropical, FundaÅao Tropical de Pesquisas
Entry Microorganisms
1
5
6
7
´
Andre Tosello) and tested for a fluorogenic reaction us-
1
2
3
4
5
6
7
8
9
Aspergillus niger CCT 4648
–
–
–
–
–
–
–
–
–
–
ing ketones 1 and 5–7 as fluorescent probes in a micro-
titer-plate set-up. A relatively slow but steady fluoro-
genic reaction was observed upon continuous monitor-
ing of the reaction at 308C over a 24-h incubation time
in half of the 60 microorganism/substrate combinations
tested (Figure 1, Table 1). When the fluorescence did
not develop, revealing an absence of BVMO, a second
set of experiments was performed incubating the experi-
ment for 24 h, after which BSA was added and the fluo- 10
rescence was measured for 24 h. The preincubation pe-
riod was introduced to detect the inducible BVMO
which was present in 5 microorganisms (Table 1, en-
Aspergillus niger CCT 5559
Aspergillus oryzae CCT 0975
Aspergillus terreus CCT 3320
13 40
–
–
–
–
Cunnighamela echinulata CCT 4259 13 120 90 70
Curvularia eragrostidis CCT 5634[b] 3 110 16
–
–
–
–
–
Curvularia lunata CCT 5628
Curvularia lunata CCT 5629
Curvularia pallescens CCT 5654[b]
Geotrichum candidum CCT 1205
Fungi CCT 5560[b]
–
–
–
–
9
–
–
7 230
4 110
5
–
11
12
17 29
30
5 260
–
–
Fungi CCT 5632[b]
–
10
–
13
14
Fungi CCT 5661
12
Rhizopus oryzae CCT 4964[b]
7
65 35 27
tries 6, 9, 11, 12 and 14).
15
Trichosporon cutaneum CCT 1903 16 230 350 290
The fluorescence assays were compared with GC/MS
monitoring of the BVMO activity on 3-hexyl-cyclobuta-
[a]
The fluorescence intensity difference (lex ¼365 nm, lem
¼
none 19 and 4-methyl-cyclohexanone 20 (Scheme 3).[19]
A Baeyer–Villiger oxidation of 19 to 19a (Scheme 3 , Ta-
ble 2) was observed with Cunnighamela echinulata CCT
4259, Curvularia lunata CCT 5629, Curvularia eragrosti-
dis CCT 5634, Curvularia pallescence CCT 5654 and As-
pergillus niger CCT 5559, in agreement with the fluores-
cence assay (Table 1). However 4-methylcyclohexanone
20 was converted to 20a (Scheme 3, Table 2) by Asper-
460 nm) between each microorganism reaction and the re-
ference (substrateþbufferþBSA) after 24 h incubation is
given in arbitrary units. There was no detectable fluores-
cence of the microorganism cultures. Conditions: 100 mM
substrate (diluted from a 2 mM stock solution in 1: 1 acet-
onitrile/water), 0.5 mg mLꢀ1 of microbial cells, 20 mM bo-
rate pH 8.8, 2 mg mLꢀ1 BSA, 308C. The above listed re-
sults are the mean values of duplicate and triplicate reac-
tions.
[b]
gillus oryzae CCT 0975 and Geotrichum candidum
CCT 1205, but these microorganisms did not convert
With preincubation with substrate at 288C for 24 h.
our fluorogenic substrates (A. oryzae) or convert very
little (G. candidum). This might be assigned to: 1) unfav- BVMO assay); 2) an oxidation with migration of the
orable reaction conditions in the microtiter plate format less substituted carbon leading to the formation of the
(24 h standing vs. 72–90 h in shaker in the classical chemically disfavored regioisomer, which is not fluoro-
genic; 3) the particular substrate selectivity of the
BVMO produced by these microorganisms; 4) a com-
peting reduction of the ketone 1, 5–7 to the correspond-
ing alcohols taking place preferentially to the BVoxida-
tion. Reduction of ketone 6 to the alcohol was indeed
observed by HPLC/UV in the microbial reaction with
Emericela nidulans CCT 3119 and C. eragrostidis CCT
5634 (data no shown). In the course of these microbial
reactions 19 and 20 were also in part reduced to secon-
dary alcohols, 19b and 20b (Scheme 3, Table 2). The al-
cohols were the only products formed with microbial
culture of Aspergillus niger CCT 4648 and Aspergillus
terreus CCT 3320,[19] for which also no fluorescence reac-
tion was observed with probes 1 and 5–7.
Monooxygenase Activity
The strongest fluorescence signal with ketones 1 and 5–
7 was produced by Trichosporon cutaneum CCT 1903,
Figure 1. Whole-cell fluorescence screening of monooxyge-
which was consistent with previous observations.[20]
nase activity with 2-couramyloxycyclopentanone 5. Condi-
tions: 100 mM 5, 0.5 mg mLꢀ1 cells, 2 mg mLꢀ1 BSA,
20 mM aqueous borate buffer pH 8.8, 308C, lex ¼365 nm,
lem ¼460 nm. See also data in Table 1.
Monitoring of the reaction over 24 h by HPLC/UV indi-
cated the direct formation of umbelliferone from these
substrates without any detectable intermediates. Al-
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Renaud Sicard et al.
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cating the presence of monooxygenases.[19] In nature
these enzymes are responsible for double bond epoxida-
tion and hydroxylation of activated methine or methyl-
ene groups similar to those present in the florescent
probes 1 and 5–7. The presence of a monooxygenase
with hydroxylating properties in T. cutaneum was estab-
lished by converting camphor to 6-hydroxycamphor
which was confirmed by GC/MS and NMR spectrosco-
py.[21] The fluorogenic reaction observed with T. cuta-
neum and ketones 1 and 5–7 is therefore best ascribed
to a direct hydroxylation of the ether methylene group
to form an unstable hemiacetal which then gives umbel-
liferone 4 (Scheme 3). The hydroxylation of the fluoro-
genic substrates such as the methyl ether of umbellifer-
one 21 is an expected hydroxylase biocatalytic reaction
at an activated methylene carbon.
Esterase Assay with Fluorogenic and Chromogenic
Lactone Substrates
The use of lactones 2, 10, 13, 14 as spectrophotometric
probes for lipases and esterase was investigated next.
A spectrophotometric lactonase assay has been describ-
ed based on 3,4-dihydrocoumarin hydrolysis (UV
270 nm),[22] but no fluorogenic or chromogenic lactone
substrates have been described thus far. The lactones
were conditioned as 10 mM stock solutions in dimethyl
sulfoxide (DMSO) and assayed for their reactivity in
aqueous buffer in the presence of 10% v/v DMSO as co-
solvent. The spontaneous hydrolysis with these lactone
substrates was much higher than previously observed
with the corresponding acyclic acyloxymethyl ethers.
The seven-membered ring lactones were significantly
more stable than the six-membered rings, whereas the
nitrophenol derived lactone 13 was particularly unsta-
ble.
Assays were conducted under neutral conditions in
phosphate buffer saline pH 7.4 to keep the background
hydrolysis at a low level. Under these conditions, a pos-
itive catalysis signal was obtained in the presence of sev-
eral lipases and esterases, with pig liver esterase samples
showing the strongest reactivity (Figure 2, Table 3)
which is in agreement of Rousseauꢁs study.[23] The
seven-membered ring umbelliferone-derived lactone 2
gave a useful signal with all enzymes, as indicated by
the value of Vrel, which measures the apparent signal
over background. This high specific reactivity was due
Scheme 3. Substrates used in detecting monooxygenases ac-
tivity.
Table 2. Biocatalytic products using 3-hexylcyclobutanone 19
and 4-methylcyclohexanone 20.[a]
Microorganisms
19a 19b 20a 20b
Aspergillus niger CCT 4648
Aspergillus niger CCT 5559
Aspergillus oryzae CCT 0975
Aspergillus terreus CCT 3320
–
80
–
–
–
–
–
–
1
54
–
–
2
–
3
1
25
23
23
93
81
75
51
94
97
–
–
Cunnighamela echinulata CCT 4259 80
–
Curvularia eragrostidis CCT 5634
Curvularia lunata CCT 5628
Curvularia lunata CCT 5629
Curvularia pallescens CCT 5654
Geotrichum candidum CCT 1205
20
44
80
54
–
10
26
–
–
–
100
[a]
Biocatalytic reactions: ketones 19–20 (approximately
20 mg each) and wet fungal biomass (2.0 g) were mixed
in Erlenmeyer flasks (125 mL) containing 30 mL of buffer
phosphate solution pH 7.0. The mixture was stirred on a
rotary shaker (288C, 140 rpm) and monitored by GC/MS
(Ref.[19]).
though the intermediate lactones of the putative Baeyer– to the very low background hydrolysis with this sub-
Villiger reaction might be too unstable in the medium to strate compared to the other lactone substrates tested.
be detectable, the presence of a possible BVMO was not
Enantioselectivity in the pig liver esterase-catalyzed
be confirmed by traditional biocatalytic reactions with reaction was investigated by analyzing the reaction by
several ketones. In most bioreactions the ketones were chiral phase HPLC, taking advantage of the strong aro-
reduced.
matic chromophore for detection. An excellent chiral
The key to this apparent enigma came from a biocata- separation was obtained for each of the four lactones
lytic experiment in which T. cutaneum was responsible on a Chiralcel-OD-H (Figure 3). Application of the ki-
for the epoxidation of the cis-jasmone double bond indi- netic resolution equation of Sih to product formation al-
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Fluorescence-Based Assay for Baeyer–Villiger Monooxygenases
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Conclusion
2-Coumaryloxy ketones 1 and 5–7 were prepared in one
step from the corresponding commercially available 2-
chloro ketones. These easily accessible substrates serve
as useful fluorogenic probes for monooxygenases under
direct culture conditions. The probes were validated
against cultures of 15 microorganisms and the results
were confirmed by GC/MS assays with 3-hexylcyclobu-
tanone 19 and 4-methylcyclohexanone 20 as substrates.
Our results indicate the above mentioned probes 1 and
5–7 are excellent for screening monooxygenases, either
hydroxylases or BVMO. Some fungi (entries 11–14, Ta-
ble 1) isolated in our laboratory[25] display interesting
monooxygenase activities and will be further investigat-
ed. The lactones 2 and 10 produced by Baeyer–Villiger
oxidation of 1 and 5, respectively, and the corresponding
nitrophenoxy-substituted lactones 13 and 14 serve as
spectroscopic chiral probes for esterases, with a par-
ticularly strong reactivity with pig liver esterase samples.
The seven-membered ring coumarin-derived lactone 2
is particularly useful due to a very low background hy-
drolysis. Enantioselectivity can be estimated by chiral-
phase HPLC of the unreacted substrates.
Figure 2. Esterase fluorescence assay with fluorogenic lac-
~
tones 2 and 10. ( ) 100 mM 2 in aqueous PꢀB1S, 10% v/v
~
DMSO, 358C; ( ) 100 mM 2 with 10 mg mL chirazyme
ꢀ1
&
E1; ( ) 100 mM 2 with 10 mg mL
chirazyme E2; (ꢀ)
100 mM 10 in aqueous PBS, 10% v/v DMSO, 358C; (þ)
100 mM 10 with 10 mg mLꢀ1 chirazyme E1; (x) 100 mM 10
with 10 mg mLꢀ1 chirazyme E2.
lowed the determination of enantioselectivity E in each
case.[24] Both samples of pig liver esterases gave enantio-
selectivities in the range between 2.3 > E > 6.6 (Ta-
ble 4).
Experimental Section
Reagents were purchased from Fluka, Aldrich, Sigma or Ac-
ros. Solvents were distilled from technical solvents. All reac-
tions were followed by TLC on Alugram SIL G/UV254 silica
Table 3. Rates of hydrolysis of lactone substrates with different enzymes.
Name of enzymes
Vobs, nM sꢀ1 (Vrel)
10
2
13
8.0
13
29
11
14
0.9
2.4
40 (44)
5.4 (5)
1.8
1.9
49 (54)
2.0
No enzyme
1.9
7.5
35 (17)
7.1
7.3
6.2
35 (17)
7.9
6.9
0.1
Pseudomonas sp. Lipase[a]
Pig liver esterase 1[b]
1.6 (15)
53 (530)
1.6 (15)
1.9 (18)
1.5 (14)
51 (510)
1.9 (18)
1.7 (16)
1.6 (16)
Candida antarctica lipase[c]
Thermomyces lanuginosa lipase[d]
Pig pancreas lipase[e]
8.8
11
32
13
12
12
Pig liver esterase 2[f]
Mucor miehei lipase[g]
Mucor miehei esterase[h]
Saccharomyces cerevisiae esterasei)
2.0
1.8
7.2
Conditions: 100 mM substrate in aqueous PBS pH 7.4, 10% v/v DMSO, 358C, 10 mg mLꢀ1 enzyme. Vrel ¼(Vobs/VnoE )ꢀ1 is in-
dicated in brackets when Vrel >5.
Enzyme sources:
[a]
Roche Chirazyme L6.
Chirazyme E1.
Chirazyme L5.
Chirazyme L8.
Chirazyme L7.
Chirazyme E2.
Chirazyme L9.
Fluka F46059.
[b]
[c]
[d]
[e]
[f]
[g]
[h]
[i]
Fluka F46069.
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Renaud Sicard et al.
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Figure 3. Chiral-phase HPLC analysis of the reaction of lactone 2 with pig liver esterase (chirazyme E1). Main plot: HPLC-
trace after 25 min and 75% of conversion. reaction. Insert: control without enzyme after 25 min. Conditions: Conditions:
100 mM substrate in aqueous PBS pH 7.4, 10% v/v DMSO, 10 mg mLꢀ1 enzyme, 258C. HPLC analysis was performed after
extraction with AcOEt using a Chiracell OD-H 0.4ꢁ22 cm column, flow rate 0.5 mL minꢀ1 2-propanol/hexane 15/85 (* solvent
peak).
Table 4. Enantioselectivities of pig liver esterase samples determined by chiral-phase HPLC analysis of the fluorogenic and
chromogenic reactions of lactone substrates.
Substrates
tR1[a] [min]
tR2[b] [min]
E (chirazyme E1)[c]
E(Chirazyme E2)[c]
10
2
13
14
88.9
60.4
39.2
31.4
107.2
68.4
60.8
42.5
2.3ꢂ0.5
6.6ꢂ0.2
4.0ꢂ1.5
3.5ꢂ1.0
2.3ꢂ0.5
5.6ꢂ1.1
2.7ꢂ0.7
4.5ꢂ1.3
Conditions: 100 mM substrate in aqueous PBS pH 7.4, 10% v/v DMSO, 10 mg mLꢀ1 enzyme, 258C. Aliquots were extracted
with AcOEt at fixed time intervals and analyzed by HPLC. Each datapoint is the average of at least three different
HPLC-analyses.
[a]
Retention time of first lactone enantiomer.
Retention time of second lactone enantiomer. HPLC conditions see Figure 3.
[b]
[c]
Enantioselectivity E was calculated in each case according to the equation: E¼ln[(1ꢀc)(1ꢀee)]/ln[(1ꢀc)(1þee)], where
c¼conversion of substrate to product and ee is the enantiomeric purity of unreacted substrate.
gel sheets (Macherey-Nagel) with detection by UV (245 and
365 nm) or with reagents like 10% ninhydrin solution in 90%
EtOH. All chromatographies (flash) were performed with Flu-
ka Silica gel 60 (0.04–0.063 mm, 230–400 mesh ASTM). Melt-
ing points were determined on a Bꢂchi 510 apparatus and are
not corrected. NMR spectra were recorded on a Bruker AC
300 for 1H (300 MHz) or C13 (75 MHz) measurements or on a
Bruker AC 400 for 1H (400 MHz) or C13 (100 MHz) measure-
ments. Chemical shifts d are given in ppm and coupling con-
stants J in Hertz. Mass spectra and accurate mass were provid-
ed by the “Service of Mass Spectrometry” of the Department
of Chemistry and Biochemistry.
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7-(2-Oxocyclohexyloxy)-2H-chromen-2-one (1)
7-(2-Oxopropoxy)-chromen-2-one (6)
Umbelliferone (0.65 g, 4 mmol) was dissolved in acetone
(15 mL). After addition of 2-chlorocyclohexanone (1.32 g,
10 mmol, 2.5 equivs.), K2CO3 (0.94 g, 6 mmol, 1.5 equivs.)
and NaI (0.9 g, 6 mmol, 1.5 equivs.) at 08C, the mixture was
stirred at 608C overnight. After the reaction was complete,
the mixture was filtered and the filtrate was evaporated. The
residue was purified by column chromatography (EtOAc/hex-
ane, 1:1) to afford 1 as a colorless solid; yield: 0.6 g (2.3 mmol,
Umbelliferone (0.65 g, 4 mmol) was dissolved in chloroace-
tone (6 mL). After addition of potassium carbonate (0.94 g,
6 mmol, 1.5 equivs.) and sodium iodide (0.9 g, 6 mmol,
1.5 equivs.) at 08C, the mixture was stirred at 258C for 4 hours.
After the reaction was complete water (20 mL) was added.
Aqueous work-up (AcOEt/brine), drying (MgSO4), evapora-
tion of the solvent, and recrystallization (acetone/hexane)
gave 6 as a colorless solid; yield: 0.605 mg (2.7 mmol, 70%);
mp 107–1098C; 1H NMR (CDCl3, 300 MHz): d¼7.64 (d, J¼
9.6 Hz, 1 H), 7.41 (d, J¼8.7 Hz, 1 H), 6.86 (dd, J¼2.7 Hz and
8.7 Hz, 1 H), 6.75 (d, J¼2.7 Hz, 1 H), 6.27 (d, J¼9.6 Hz,
1 H), 4.64 (s, 2 H),2.30 (s, 3 H); 13C NMR (CDCl3, 75 MHz):
d¼204.2, 161.3, 160.2, 156.4, 143.8, 129.8, 114.6, 114.0, 113.3,
102.5, 73.6, 27.3; EI-MS: m/z¼218 [M]þ; HR-MS-EI: calcd.
for C13H12O4: 218.0579; m/z¼218.0578.
1
58%); mp 167–1698C; Rf ¼0.4; H NMR (CDCl3, 300 MHz):
d¼7.63 (d, J¼9.5 Hz, 1 H), 7.37 (d, J¼8.6 Hz, 1 H), 6.83
(dd, J¼2.4 Hz and 8.6 Hz, 1 H), 6.70 (d, J¼2.4 Hz, 1 H), 6.25
(d, J¼9.6 Hz, 1 H), 4.75 (dd, J¼10.1 Hz and 5.4 Hz, 1 H),
2.62 (m, 1 H), 2.43 (m, 2 H), 2.07 (m, 3 H),1.81 (m, 2 H); 13C
NMR (CDCl3, 75 MHz): d¼207.1, 161.7, 161.4, 156.3, 143.9,
129.5, 114.1, 113.8, 113.7, 103.1, 81.4, 41.3, 34.9, 28.3, 23.7; EI-
MS: m/z¼258 [M]þ; HR-MS-EI: calcd. for C15H14O4:
258.0892; m/z¼258.0892.
7-(1-Methyl-2-oxopropoxy)-chromen-2-one (7)
Umbelliferone (0.65 g, 4 mmol) was dissolved in acetone
(10 mL). After addition of 3-chloro-2-butanone (1 mL,
10 mmol, 2.5 equivs.), K2CO3 (0.94 g, 6 mmol, 1.5 equivs.)
and NaI (0.9 g, 6 mmol, 1.5 equivs.) at 08C, the mixture was
stirred at 608C overnight. After the reaction was complete,
the mixture was filtered and the filtrate was evaporated to af-
ford 7 as a colorless solid; yield: 0.9 g (3.88 mmol, 98%); mp
86–888C; 1H NMR (CDCl3, 300 MHz): d¼7.63 (d, J¼
9.4 Hz, 1 H), 7.38 (d, J¼8.7 Hz, 1 H), 6.81 (dd, J¼2.4 Hz and
8.7 Hz, 1 H), 6.73 (d, J¼2.4 Hz, 1 H), 6.27 (d, J¼9.6 Hz,
1 H), 4.70 (q, J¼7.8 Hz, 1 H), 2.20 (s, 3 H),1.55 (d, J¼7.8 Hz,
3 H); 13C NMR (CDCl3, 75 MHz): d¼208.9, 161.5, 161.0,
156.4, 143.8, 129.8, 114.4, 113.9, 113.2, 103.0, 80.1, 25.5, 18.0;
EI-MS: m/z¼232 [M]þ; HR-MS-EI: calcd. for C13H12O4:
232.0735; m/z¼232.0735.
7-(7-Oxooxepan-2-yloxy)-2H-chromen-2-one (2)
Ketone 1 (150 mg, 0.58 mmol) was dissolved in dichlorome-
thane (2 mL). After addition of m-CPBA (186 mg,
0.76 mmol, 1.3 equivs.) and NaHCO3 (64 mg, 0.76 mmol,
1.3 equivs.) at 08C, the mixture was stirred at 258C during 3
hours. Aqueous work-up (CH2Cl2/water/brine), drying
(MgSO4), evaporation of the solvent and purification by col-
umn chromatography (hexane/ethyl acetate, 6:4, Rf ¼0.2)
gave 2 as colorless solid; yield: 110 mg (0.40 mmol, 69%); mp
116–1188C; 1H NMR (CDCl3, 300 MHz): d¼7.64 (d, J¼
9.6 Hz, 1 H), 7.41 (d, J¼9.3 Hz, 1 H), 7.01 (m, 2 H), 6.29 (d,
J¼9.6 Hz, 1 H), 5.92 (d, J¼5.4 Hz, 1 H), 2.75 (m, 2 H),
1.74–2.34 (m, 5 H); 13C NMR (CDCl3, 75 MHz): 173.4, 161.3,
159.3, 156.1, 143.7, 129.8, 115.3, 115.1, 113.9, 105.1, 98.9, 37.4,
34.4, 23.7, 23.5; EI-MS: m/z¼274 [M]þ; HR-MS-EI: calcd.
for C15H14O5: 274.0841; m/z¼274.0839.
2-(4-Nitrophenoxy)-cyclopentanone (8)
4-Nitrophenol (0.46 g, 3.3 mmol) was dissolved in acetone
(15 mL). After addition of 2-chlorocyclopentanone (1 mL,
10 mmol, 3 equivs.), potassium carbonate (1.03 g, 6.6 mmol,
2 equivs.) and sodium iodide (0.99 g, 6.6 mmol, 2 equivs.) at
08C, the mixture was stirred at 608C overnight. After the reac-
tion was complete, the mixture was filtered and the filtrate was
evaporated. Aqueous work-up (AcOEt/NH4Cl, then water,
then brine), evaporation of the organic phase and column chro-
matography (EtOAc/hexane, 2:8, Rf ¼0.3) gave 8 as a dark yel-
low solid; yield: 0.45 g (3 mmol, 69%); mp 70–738C; 1H NMR
(CDCl3, 300 MHz): d¼8.11 (d, J¼9.2 Hz, 2 H), 6.98 (d, J¼
9.2 Hz, 2 H), 4.74 (m, 1 H), 2.50 (m, 1 H), 2.36 (m, 2 H), 2.13
(m, 1 H),1.95 (m, 2 H); 13C NMR (CDCl3, 75 MHz): d¼
213.3, 163.6, 126.5, 116.1, 80.1, 35.9, 30.1, 17.7; EI-MS: m/z¼
221 [M]þ; HR-MS-EI: calcd. for C11H11NO4: 221.0688; m/z¼
221.0688.
7-(2-Oxocyclopentyloxy)-2H-chromen-2-one (5)
Umbelliferone (0.65 g, 4 mmol) was dissolved in acetone
(15 mL). After addition of 2-chlorocyclopentanone (1 mL,
10 mmol, 2.5 equiv.), K2CO3 (0.94 g, 6 mmol, 1.5 equivs.) and
NaI (0.9 g, 6 mmol, 1.5 equivs.) at 08C, the mixture was stirred
at 608C overnight. After the reaction was complete, the mix-
ture was filtered and the filtrate was evaporated. The residue
was purified by column chromatography (EtOAc/hexane,
4:6) to afford 5 as a colorless solid; yield: 0.6 g (2.5 mmol,
1
62%); mp 125–1278C; Rf ¼0.3; H NMR (CDCl3, 300 MHz):
d¼7.64 (d, J¼9.5 Hz, 1 H), 7.38 (d, J¼8.3 Hz, 1 H), , 6.94
(dd, J¼2.6 Hz and 8.3 Hz, 1 H), 6.90 (d, J¼2.4 Hz, 1 H), 6.28
(d, J¼9.4 Hz, 1 H), 4.68 (m, 1 H), 2.43 (m, 1 H), 2.40 (m,
2 H), 2.07 (m, 1 H), 2.05 (m, 2 H); 13C NMR (CDCl3,
75 MHz): d¼213.5, 161.7, 161.7, 156.3, 144.0, 129.5, 114.3,
114.1, 113.9, 103.4, 80.2, 34.9, 30.0, 17.9; EI-MS: m/z¼244
[M]þ; HR-MS-EI: calcd. for C14H12O4: 244.0735, m/z¼
244.0733.
2-(4-Nitrophenoxy)-cyclohexanone (9)
4-Nitrophenol (0.46 g, 3.3 mmol) was dissolved in acetone
(15 mL). After addition of 2-chlorocyclohexanone (1.14 mL,
10 mmol, 3 equivs.), K2CO3 (1.03 g, 6.6 mmol, 2 equivs.) and
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Renaud Sicard et al.
FULL PAPERS
NaI (0.99 g, 6.6 mmol, 2 equivs.) at 08C, the mixture was stirred
at 608C overnight. After the reaction was complete, the mix-
ture was filtered and the filtrate was evaporated. Aqueous
work-up (AcOEt/NH4Cl, then water, then brine), evaporation
of the organic phase and recrystallization (acetone/hexane)
gave 9 as a colorless solid; yield: 0.71 g (3 mmol, 92%); mp
90–928C; 1H NMR (CDCl3, 300 MHz): d¼8.15 (d, J¼
9.2 Hz, 2 H), 6.87 (d, J¼9.2 Hz, 2 H), 4.77 (m, 1 H), 2.5 (m,
1 H), 2.41 (m, 2 H), 2.07 (m, 3 H), 1.80 (m, 2 H); 13C NMR
(CDCl3, 75 MHz): d¼206.9, 163.4, 126.5, 115.8, 81.4, 41.3,
34.9, 28.2, 23.7; EI-MS: m/z¼235 [M]þ; HR-MS-EI: calcd.
for C12H13NO4: 235.0845; m/z¼235.0844.
of the solvent and purification by column chromatography
(hexane/ethyl acetate, 6:4, Rf ¼0.3) gave 12 as a colorless solid;
yield: 170 mg (0.69 mmol, 80%); mp 80–838C; 1H NMR
(CDCl3, 300 MHz): d¼7.62 (d, J¼9.6 Hz, 1 H), 7.37 (d, J¼
8.5 Hz, 1 H), 6.84 (dd, J¼2.4 Hz and 8.5 Hz, 1 H), 6.82 (d,
J¼2.4 Hz, 1 H), 6.56 (q, J¼5.1 Hz, 1 H), 6.24 (d, J¼9.4 Hz,
1 H), 2.04 (s, 3 H),1.60 (d, J¼5.1 Hz, 3 H); 13C NMR (CDCl3,
75 MHz): d¼170.4, 161.5, 159.5, 156.2, 143.8, 129.7, 115.0,
114.7, 114.1, 104.8, 93.7, 21.7, 21.1; EI-MS: m/z¼248 [M]þ;
HR-MS-EI: calcd. for C13H12O5: 248.0685; m/z¼248.0688.
6-(4-Nitrophenoxy)-tetrahydropyran-2-one (13)
Ketone 8 (0.2 g, 0.9 mmol) was dissolved in dichloromethane
(5 mL). After addition of m-CPBA (0.29 g, 1.18 mmol,
1.3 equivs.) and NaHCO3 (0.1 mg, 1.18 mmol, 1.3 equivs.) at
08C, the mixture was stirred at 258C during 3 hours. Aqueous
work-up (CH2Cl2/aqueous NH4Cl/brine), drying (MgSO4),
evaporation of the solvent and recrystallization (dichlorome-
thane/hexane) gave 13 as a colorless solid; yield: 120 mg
(0.45 mmol, 50%); mp 85–908C; 1H NMR (CDCl3,
300 MHz): d¼8.21 (d, J¼7.2 Hz, 2 H), 7.17 (d, J¼7.2 Hz,
2 H), 6.07 (m, 1 H), 2.67 (m, 2 H), 2.29 (m, 1 H), 2.15 (m,
2 H), 1.96 (m, 1 H); 13C NMR (CDCl3, 75 MHz): d¼169.9,
161.5, 143.8, 126.6, 117.2, 99.3, 30.5, 28.0, 15.8; ESI-MS: m/
z¼238 [MþH]þ; HR-MS: calcd. for C12H13NO5: 238.0715;
m/z¼238.0715.
7-(Tetrahydro-6-oxo-2H-pyran-2-yloxy)-2H-chromen-
2-one (10)
Ketone 5 (50 mg, 0.205 mmol) was dissolved in dichlorome-
thane (1 mL). After addition of m-CPBA (65.7 mg,
0.266 mmol, 1.3 equivs.) and NaHCO3 (22 mg, 0.266 mmol,
1.3 equivs.) at 08C, the mixture was stirred at 258C during 3
hours. Aqueous work-up (CH2Cl2/water/brine), drying
(MgSO4), evaporation of the solvent and purification by col-
umn chromatography (hexane/ethyl acetate, 1:1, Rf ¼0.4)
gave 10 as colorless solid; yield: 46 mg (0.177 mmol, 86%);
mp 151–1538C; 1H NMR (CDCl3, 300 MHz): d¼7.64 (d, J¼
9.6 Hz, 1 H), 7.41 (d, J¼9.3 Hz, 1 H), 7.01 (m, 2 H), 6.29 (d,
J¼9.5 Hz, 1 H), 6.05 (dd, J¼3.2 Hz and 3.7, 1 H), 2.72 (m,
2 H), 1.91–2.32 (m, 4 H); 13C NMR (CDCl3, 75 MHz): d¼
169.5, 161.4, 159.6, 156.1, 143.7, 129.7, 115.1, 115.0, 114.1,
105.2, 98.6, 30.5, 27.4, 15.9; EI-MS: m/z¼260 [M]þ; HR-MS-
EI: calcd. for C14H12O5: 260.0684; m/z¼260.0683.
7-(4-Nitrophenoxy)oxepan-2-one (14)
Ketone 9 (0.5 g, 2.13 mmol) was dissolved in dichloromethane
(15 mL). After addition of m-CPBA (0.68 g, 2.76 mmol,
1.3 equivs.) and NaHCO3 (0.23 mg, 2.76 mmol, 1.3 equivs.) at
08C, the mixture was stirred at 258C during 3 hours. Aqueous
work-up (CH2Cl2/aqueous NH4Cl/brine), drying (MgSO4),
evaporation of the solvent and recrystallization (acetone/hex-
ane) gave 14 as a colorless solid; yield: 360 mg (1.5 mmol, 67%);
mp 112–1178C; 1H NMR (CDCl3, 300 MHz): d¼8.22 (d, J¼
7.1 Hz, 2 H), 7.17 (d, J¼7.1 Hz, 2 H), 5.93 (d, J¼4.7 Hz,
1 H), 2.73 (m, 2 H), 2.36 (m, 1 H), 2.16 (m, 2 H), 1.59–1.96
(m, 3 H); 13C NMR (CDCl3, 75 MHz): d¼173.2, 161.1, 143.9,
126.6, 117.1, 98.6, 37.4, 34.3, 23.5, 23.4; EI-MS: m/z¼251
[M]þ; HR-MS-EI: calcd. for C12H13NO5: 251.0796; m/z¼
251.0794.
Acetic Acid 1-(2-Oxo-2H-chromen-7-yloxy)-methyl
Ester (11) by Oxidation of Ketone 1
Ketone 6 (300 mg, 1.37 mmol) was dissolved in dichlorome-
thane (20 mL). After addition of m-CPBA(714 mg,
2.06 mmol, 1.5 equivs.) and NaHCO3 (150 mg, 1.78 mmol,
1.3 equivs.) at 08C, the mixture was stirred at 258C overnight.
Aqueous work-up (CH2Cl2/aqueous NH4Cl/brine), drying
(MgSO4), evaporation of the solvent and recrystallization of
the residue (acetone/hexane) gave 11 as a colorless solid; yield:
260 mg (1.11 mmol, 81%); mp 161–1658C; Rf ¼0.3 (hexane/
1
ethyl acetate, 1:1); H NMR (CDCl3, 300 MHz): d¼7.64 (d,
J¼9.6 Hz, 1 H), 7.42 (d, J¼8.4 Hz, 1 H), 7.02 (d, J¼2.4 Hz,
1 H), 6.95 (dd, J¼2.4 Hz and 8.4 Hz, 1 H), 6.32 (d, J¼9.6 Hz,
1 H), 5.80 (s, 2 H), 2.14 (s, 3 H); 13C NMR (CDCl3, 75 MHz):
d¼170.3, 161.5, 160.3, 156.2, 143.8, 129.7, 115.0, 114.7, 114.2,
104.1, 85.4, 21.7; ESI-MS: m/z¼235 [MþH]þ; calcd. for
C12H10O5: 235.0606; m/z¼235.0611.
Monooxygenase Assays
Microorganisms: Yeast and fungi were cultured for 3–4 days, at
288C, in test tubes (h¼18 cm; ø¼1.5 cm) containing ca. 10 mL
of yeast-malt-agar (YMA) medium (3 g Lꢀ1 yeast extract
Merck, 3 g Lꢀ1 malt extract Merck, 5 g Lꢀ1 peptone Difco,
10 g Lꢀ1 glucose, 20 g Lꢀ1 agar Merck, H2O) and 10 mL of
malt agar (MA) medium (20 g Lꢀ1 malt extract Merck, 20 g
Lꢀ1 agar Merck, H2O), respectively. Just prior to the tests the
grown colonies were weighed into 1.5 mL Eppendorf tubes
and suspended in borate buffer in order to obtain 1 mg of
wet cells per mL.
Acetic Acid 1-(2-Oxo-2H-chromen-7-yloxy)-ethyl
Ester (12)
Ketone 7 (200 mg, 0.86 mmol) was dissolved in dichlorome-
thane (5 mL). After addition of m-CPBA (223 mg, 1.3 mmol,
1.5 equivs.) and NaHCO3 (74 mg, 1.1 mmol, 1.3 equivs.) at
08C, the mixture was stirred at 258C during 10 hours. Aqueous
work-up (CH2Cl2/water/brine), drying (MgSO4), evaporation
Fluorescence measurements without preincubation: All buf-
fers and solutions were prepared using deionized H2O. Micro-
bial cells were diluted from 1 mg mLꢀ1 suspensions in 20 mmol
1048
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Fluorescence-Based Assay for Baeyer–Villiger Monooxygenases
FULL PAPERS
Lꢀ1 borate buffer (pH 8.8; 100 mL). Substrates were diluted
from 2 mmol Lꢀ1 stock solutions in 50% aqueous MeCN,
(10 mL). BSA was diluted from a 4.4 mg mLꢀ1 stock solution
in 20 mmol Lꢀ1 borate (pH 8.8; 90 mL). The 200 mL assays
were followed in individual wells of flat-bottom polypropylene
96-well microtiter plates (Costar) with a Fluoroskan Ascent flu-
orescence plate reader (Labsystems, filters lex 365ꢂ20 nm, lem
460ꢂ20 nm) (Table 1, entries 1–5, 7, 8, 10, 13, 15)
References
[1] a) B. Marrs, S. Delagrave, D. Murphy, Curr. Opin. Micro-
biol. 1999, 2, 241–245; b) W. P. C. Stemmer, Proc. Natl.
Acad. Sci. USA 1994, 91, 10747–10751; c) W. P. C.
Stemmer, Nature 1994, 370, 389–391; d) L. You, F. H.
Arnold, Protein. Eng. 1996, 9, 77–83; e) M. T. Reetz,
K.-E. Jaeger, Chem. Eur. J. 2000, 6, 407–412; f) S. V. Tay-
lor, P. Kast, D. Hilvert, Angew. Chem. 2001, 113, 3408–
3436; Angew. Chem. Int. Ed. 2001, 40, 3310–3335.
[2] a) Enzyme Assays: A Practical Approach. (Eds.: R. Ei-
senthal, M. Danson), Oxford University Press, Oxford,
2002; b) Enzyme Assays: Essential Data. (Eds.: S. Gul,
S. K. Sreedharan, K. Brocklehurst), John Wiley & Sons,
Chicester, 1998; c) M. T. Reetz, Angew. Chem. 2001,
113, 292–320; Angew. Chem. Int. Ed. 2001, 40, 284–
310; d) D. Wahler, J.-L. Reymond, Curr. Opin. Chem.
Biol. 2001, 5, 152–158; e) D. Wahler, J.-L. Reymond,
Curr. Opin. Biotechnol. 2001, 12, 535–544; f) J.-P. God-
dard, J.-L. Reymond, Trends Biotechnol. 2004, 22, 363–
370; g) J.-P. Goddard, J.-L. Reymond, Curr. Opin. Bio-
technol. 2004, 15, 314–322.
Fluorescence measurements with preincubation: These ex-
periments were performed following the above described pro-
tocol without BSA and incubating the microorganisms in the
microtiter plate at 288C for 24 h.. Fluorescence was monitored
for 24 h after BSA addition. (Table 1, entries 6, 9, 11, 12, 14).
Esterase Assays
Kinetic measurements: All buffers and solutions were prepared
using MilliQ deionized water, the pH being adjusted with
aqueous NaOH and aqueous HCl solutions. Enzymes were
used as 1 mg mLꢀ1 stock solutions in PBS (10 mM phosphate,
150 mM NaCl, pH 7.4) and preserved at 48C. Stock solutions of
substrates (10 mM and 1 mM) were prepared in DMSO and
stored at ꢀ208C. Assays (0.2 mL for fluorogenic substrates
and 0.1 mL for chromogenic substrates) were followed in
individual wells of round-bottom polypropylene 96-well-plates
(Costar) with Cytofluor II Fluorescence Plate Reader (Persep-
tive Biosystems, filters lex ¼360ꢂ20, lem ¼460ꢂ20 nm) for flu-
orogenic substrates and in individual wells of round-bottom
polyprostyrene 96-well-plates (Costar) with Spectramax 250
Microplate Spectrophotometer (Molecular Devices) for chro-
mogenic substrates. The rates indicated in Table 2 are derived
from the steepest linear portion of each curve. Conditions of as-
say: to a solution (0.1 or 0.05 mL) of enzyme at 0.02 mg mLꢀ1
(0.01 mg mLꢀ1 final concentration), is added 0.1 or 0.05 mL
of a substrates solution at 0.2 mmol Lꢀ1 (0.1 mM final concen-
tration) containing 20% of DMSO (10% final concentration).
Determination of enantioselectivity: Reactions were fol-
lowed in Eppendorf tubes (1 mL). For measurement set, a
stock solution of substrates at 1 mM in DMSO were prepared
and mixed to the stock solution of enzyme. Then an extraction
using EtOAc was done just before the measurement by HPLC.
Reaction conditions: 100 mM substrate, 10% DMSO, incuba-
tion with enzyme (10 mg mLꢀ1) at room temperature with dif-
ferent reaction time(5, 10 and 15 min). Experiments were done
on column Chiralcel OD-H (0.46ꢁ25 cm), detection by UV
[3] a) G. Klein, J.-L. Reymond, Bioorg. Med. Chem. Lett.
1998, 8, 1113; b) G. Klein, J.-L. Reymond, Helv. Chim.
Acta 1999, 82, 400.
[4] a) N. Jourdain, R. Perez-Carlon, J.-L. Reymond, Tetrahe-
´
`
dron Lett. 1998, 39, 9415; b) R. Perez Carlon, N. Jour-
dain, J.-L. Reymond, Chem. Eur. J. 2000, 6, 4154.
[5] a) F. Badalassi, D. Wahler, G. Klein, P. Crotti, J.-L. Rey-
mond, Angew. Chem. Int. Ed. 2000, 39, 4067–4070;
b) E. M. Gonzalez-Garcia, J. Grognux, D. Wahler, J.-L.
Reymond, Helv. Chim. Acta 2003, 86, 2458–2470; c) E.
Nyfeler, J. Grognux, D. Wahler, J.-L. Reymond, Helv.
Chim. Acta 2003, 86, 2919–2927.
[6] F. Badalassi, P. Crotti, J.-L. Reymond, Helv. Chim. Acta
2002, 82, 3090.
[7] E. Leroy, N. Bensel, J.-L. Reymond, Adv. Syn. Catal.
2003, 345, 859–865.
[8] F. Badalassi, G. Klein, P. Crotti, J.-L. Reymond, Eur. J.
Org. Chem. 2004, 2557–2566.
[9] E. M. Gonzalez-Garcia, V. Helaine, G. Klein, M. Schuer-
mann, G. Sprenger, W.-D. Fessner, J.-L. Reymond,
Chem. Eur. J. 2003, 9, 893.
[10] a) D. Wahler, F. Badalassi, P. Crotti, J.-L. Reymond, An-
gew. Chem. Int. Ed. 2001, 40, 4457–4460; c) D. Wahler, F.
Badalassi, P. Crotti, J.-L. Reymond, Chem. Eur. J. 2002,
8, 3211–3228; d) J.-L. Reymond, D. Wahler, F. Badalass-
si, H. K. Nguyen, Patent WO0192563, 2001-12-07; e) J.-L.
Reymond, D. Wahler ChemBioChem 2002, 3, 701–708;
f) J. Grognux, J.-L. Reymond, ChemBioChem 2004, 5,
826–831; g) J.-P. Goddard, J.-L. Reymond, J. Am.
Chem. Soc. 2004, 126, 11116–11117.
320 nm, eluent: hexane/2-propanol, 85/10; flow: 0.5 mL minꢀ1
UV 320 nm.
,
Acknowledgements
`
[11] a) D. Wahler, O. Boujard, F. Lefevre, J.-L. Reymond, Tet-
rahedron 2004, 60, 703–710; b) J. Grognux, D. Wahler, E.
Nyfeler, J.-L. Reymond, Tetrahedron: Asymmetry 2004,
15, 2981–2989.
This work was financially supported by the University of Berne,
the Swiss National Science Foundation, the COST program
´
D16, and Proteus SA, Nꢀmes, France. LSC is indebted to Con-
´
[12] A. Sevestre, V. Helaine, G. Guyot, C. Martin, L. Hec-
selho Nacional de Pesquisa (CNPq, Brazil) and AJM is indebt-
ed to CNPq and FAPESP (Brazil) for financial support.
quet, Tetrahedron Lett. 2003, 44, 827–830.
Adv. Synth. Catal. 2005, 347, 1041–1050
asc.wiley-vch.de
ꢀ 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1049

Renaud Sicard et al.
FULL PAPERS
[13] M. C. Gutierrez, A. Sleegers, H. D. Simpson, V. Alp-
hand, R. Furstoss, Org. Biomol. Chem. 2003, 1, 3500–
3506.
[14] N. Bensel, M. T. Reymond, J.-L. Reymond, Chem. Eur. J.
2001, 7, 4604.
[20] B. Bicalho, L. S. Chen, J. Grognux, J-L. Reymond, A. J.
Marsaioli, J. Braz. Chem. Soc. 2004, 15, 911–916.
[21] A. L. M. Porto. PhD thesis, 2002, Chemistry Institute,
State University of Campinas.
[22] M. Kataoka, K. Honda, S. Shimizu, Eur. J. Biochem.
[15] E. Leroy, N. Bensel and J.-L. Reymond, Bioorg. Med.
Chem. Lett. 2003, 13, 2105–2108.
2000, 267, 3–10.
[23] L. Blanco, E. Guibe-Jampel, G. Rousseau, Tetrahedron
´
[16] M. D. Mihovilovic, B. Mꢂller, P. Stanetty, Eur. J. Org.
Chem. 2002, 3711–3730.
[17] C. T. Walsh, Y.-C. J. Chen, Angew. Chem. 1988, 100,
342–352.
[18] A. B. Watts, J. Whitcher, C. S. Whitcher, J. A. Littlechild,
Biocatal. Biotrans. 2002, 20, 209–214.
[19] R. A. C. GonÅalves, A. L. M. Porto, L. Pinheiro, J. R.
Cagnon, G. P. Manfio, A. J. Marsaioli, Food Technol. Bi-
otechnol. 2004, 42, 355–361.
Lett. 1988, 29, 1915–1918.
[24] a) C-S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am.
Chem. Soc. 1982, 104, 7294–7298.; b) H. B. Kagan, J. C.
Fiaud, in: Topics in Stereochemistry; John Wiley &
Sons, New York, 1988, Vol. 18, p. 249.8.
[25] B. Bicalho, R. A. C. GonÅalves, A. P. M. Zibordi, G. P.
Manfio, A. J. Marsaioli, Z. Naturforsch. 2003, 58c, 746–
751.
1050
ꢀ 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2005, 347, 1041–1050