Baeyer-Villiger oxidations catalyzed by engineered microorganisms: Enantioselective synthesis of δ-valerolactones with functionalized chains

Article abstract of DOI:10.1139/v02-035

Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp NCIMB 9871 expressed in baker's yeast and in E. coli and cyclopentanone monooxygenase (CPMO) from Comamonas (previously Pseudomonas) sp. NCIMB 9872 expressed in E. coli are new bioreagents for Baeye

Full text of DOI:10.1139/v02-035

613
Baeyer-Villiger oxidations catalyzed by engineered
microorganisms: Enantioselective synthesis of -
valerolactones with functionalized chains
Shaozhao Wang, Gang Chen, Margaret M. Kayser, Hiroaki Iwaki, Peter C.K. Lau,
and Yoshie Hasegawa
Abstract: Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp NCIMB 9871 expressed in baker’s yeast
and in E. coli and cyclopentanone monooxygenase (CPMO) from Comamonas (previously Pseudomonas) sp. NCIMB
9872 expressed in E. coli are new bioreagents for Baeyer-Villiger oxidations. These engineered microorganisms, requir-
ing neither biochemical expertise nor equipment beyond that found in chemical laboratories, were evaluated as reagents
for Baeyer-Villiger oxidations of cyclopentanones substituted at the 2-position with polar and nonpolar chains suitable
for further modifications. Two such functionalized substrates that can be transformed into highly enantiopure lactones
were identified. The performance and the potential of these bioreagents are discussed.
Key words: enantioselective Baeyer-Villiger oxidations, biotransformations, cyclohexanone monooxygenase, cyclo-
pentanone monooxygenase, engineered baker’s yeast, recombinant E. coli, optically pure 2-substituted cyclopentanones,
optically pure lactones.
Résumé : La monooxygénase de la cyclohexanone (« CHMO ») obtenue à partir d’Acinetobacter sp NCIMB 9871 ex-
traite de la levure pâtissière et d’E. coli et la monooxygénase de la cyclopentanone (« CPMO ») de Comamonas (anté-
rieurement Pseudomonas) sp. NCIMB 9872 extraite d’E. coli sont les nouveaux bioréactifs pour les oxydations de
Baeyer-Villiger. Ces microorganismes, qui ne nécessitent ni expertise biochimique ni équipement supplémentaire par
rapport à ce que l’on retrouve dans les laboratoires chimiques, ont été évalués comme réactifs pour des oxydations de
Baeyer-Villiger de cyclopentanones substituées en position 2 par des chaînes polaires ainsi que non polaires suscepti-
bles d’être modifiées ultérieurement. On a identifié deux de ces substrats fonctionnalisés qui peuvent être éventuelle-
ment transformés en lactones de grande pureté énantiomérique. On discute de la performance et du potentiel de ces
bioréactifs.
Mots clés : oxydations de Baeyer-Villiger, biotransformations, monooxygénase de la cyclohexanone, monooxygénase de
la cyclopentanone, levure pâtissière, E. coli recombinant, cyclopentanone substituée en position 2 optiquement pure,
lactones optiquement pures.
[Traduit par la Rédaction] Wang et al. 621
Introduction
a hundred years since its discovery in 1899 by Baeyer and
Villiger (1). Many developments and changes to the reaction
occurred over the years (2), and the accepted two-step mech-
anism is familiar to chemists (2, 3). In general, this is a suc-
cessful reaction and problems only arise when scaling-up is
required because peracid oxidants are toxic and intrinsically
unstable. For this reason, new oxidizing agents continue to
be investigated and several have been introduced over the
years (4). The growing interest in the preparation of opti-
cally pure compounds has stimulated interest in creating
enantioselective Baeyer-Villiger oxidations, and a number of
transition-metal-based oxidants have been described (5). The
yields and enantiomeric excesses (ee) of the lactone prod-
ucts varied, but were typically in the order of 30–70% ee.
“Biological” Baeyer-Villiger oxidants, recognized for their
use for several decades, perform these oxidations on a broad
variety of compounds, frequently with good yields and with
superior enantioselectivity (6). Although many microorgan-
isms produce enzymes that perform Baeyer-Villiger reac-
tions, two bacterial species Acinetobacter sp. and Pseudomona,
have received the most attention in recent years. These
The transformation of cyclic ketones to lactones is most
frequently performed using peroxyacids. The lactones are
important intermediates in organic synthesis, and hence the
reaction has been studied and extensively employed for over
Received 20 November 2001. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 17 May 2002.
We would like to dedicate this paper to J. Bryan Jones whose
vision inspired us all.
S. Wang, G. Chen,1 and M.M. Kayser.2 Department of
Physical Sciences, University of New Brunswick, Saint John,
NB E2L 4L5, Canada.
H. Iwaki, and P.C.K. Lau. Biotechnology Research Institute,
N.R.C., Montreal, QC H4P 2R2, Canada.
Y. Hasegawa. Department of Biotechnology, Kansai
University,Yamate-cho, Suita, Osaka 564–8680, Japan.
1Parts of this work stem from Gang Chen’s Ph.D. thesis.
University of New Brunswick. 1999.
2Corresponding author (e-mail: kayser@unbsj.ca).
Can. J. Chem. 80: 613–621 (2002)
DOI: 10.1139/V02-035
© 2002 NRC Canada

614
Can. J. Chem. Vol. 80, 2002
Scheme 1.
O
O
O
+
R
R
Br
COOMe
COOMe
H3O
K2CO3,
acetone,
R
∆
∆
2a-f, k
g = hydroxymethyl
h = methoxymethyl
i = allyloxymethyl
1
R : a = allyl
b = n-butyl
c = cyclopentyl
d = isobutyl
j = acetoxymethyl
e = benzyl
k = ethoxycarbonylmethyl
f = 3-bromopropyl
organisms and (or) the Baeyer-Villiger enzymes (flavin
monooxygenases) that they produce are biological reagents
of real value in organic synthesis.
In an earlier paper, we reported several successful Baeyer-
Villiger oxidations of 2-alkyl cyclopentanones mediated by
the recombinant yeast (CHMO) (20). Our objective in the
current project was to evaluate the recombinant CHMO- and
CPMO-expressing strains as reagents for Baeyer-Villiger ox-
idations of 2-substituted cyclopentanones with functional
groups on the side chain. Several such substrates were pre-
pared and subjected to biotransformations. The stereo-
chemical results of this work are discussed in this paper.
Pseudomonas sp. NCIMB 10007, which carries genes in-
ducible by growth on (+)- or (–)-camphor to produce the
camphore flavin monooxygenase enzymes, has been investi-
gated in numerous reactions. The crude-enzyme extracts from
this organism were separated on the basis of the co-factor re-
quirement to give two fractions: NADH-dependent MO1 and
NADPH-dependent MO2. These fractions were used as se-
lective catalysts in reactions with various unnatural sub-
strates (6b, c, 7, 8). Elective culture grown on cyclohexanol
allowed Donoghue and Trudgill (9) to identify Acinetobacter
sp. NCIMB 9871. Since then, CHMO has been the subject of
several mechanistic and biochemical studies (10–12). Extrac-
tion of cyclohexanone monooxygenase from this strain
(CHMO EC 1.14.13.22) was optimized by Trudgill (13),
and both the microorganism and enzyme continued to be ex-
tensively investigated (see reviews listed in ref. 6). The high
enantioselectivity and broad spectrum of substrate accept-
ability made the enzyme and the organism the most widely
used bioreagents for Baeyer-Villiger oxidations. Since, how-
ever, Acinetobacter sp. NCIMB 9871 is a class 2 pathogen
and the isolated CHMO enzyme is relatively unstable and
requires the NADPH co-factor, these biotransformations
were not embraced enthusiastically by nonspecialists. The
recently constructed recombinant strains of baker’s yeast
[15C(pKR001)] and E. coli [BL21(DE3)(pMM4)] over-
expressing CHMO, which can be used to perform
enantioselective Baeyer-Villiger oxidations in ordinary shake
flasks in any organic laboratory (14, 15), should place these
versatile reagents at the disposal of all organic chemists.
Elective culture grown on cyclopentanol yielded Pseudo-
monas sp. NCIMB 9872, now identified as Comamonas
(16). This organism was able to oxidize cyclopentanol, via
cyclopentanone and δ-valerolactone, to glutarate and, pre-
sumably, to acetate by an inducible set of enzymes (17). Pu-
rified cyclopentanone monooxygenase (CPMO; EC
1.14.13.16) is NADPH-specific (17b, c). Compared with
NCIMB 9871, it has been studied relatively little in Baeyer-
Villiger oxidations of unnatural substrates, probably because
it was assumed to have stereoselectivity similar to that of
cyclohexanone monooxygenase from Acinetobacter (6b, c,
7, 18, 19). We have now cloned and overexpressed the gene
for cyclopentanone monooxygenase in the laboratory E. coli
strain DH5α, [E. coli (CPMO)] making it as accessible as E.
coli (CHMO) for development as another reagent for biolog-
ical Baeyer-Villiger oxidations (16).
Results
2-Cyclopentylcyclopentanone (2c) was purchased from
Aldrich, and compounds 2d–f were prepared in good yields
by alkylation and decarboxylation of methyl cyclopenta-
none-2-carboxylate as shown in Scheme 1. 2-Allylcyclo-
pentanone (2a) was prepared by direct alkylation of
cyclopentanone. 2-Oxocyclopentyl-1-methanol (2g) and 2-
alkoxymethylcyclopentanones 2h and 2i were synthesized in
3 and 4 steps, respectively, from methyl cyclopentanone-2-
carboxylate. In these reactions, the ketone was protected as
an ethylene ketal, and the ester group was reduced by
LiAlH4 to give hydroxyketal 4. Subsequent hydrolysis of the
ketal with a mild Lewis acid catalyst preserved the hydroxyl
group and yielded hydroxyketone 2g. Alkylation of 4 fol-
lowed by hydrolysis with a dilute aqueous acid gave
alkoxyketones 2h and 2i (Scheme 2). The synthesis of ester
2k paralleled that for simple alkyl side chains. Coupling of
ethyl cyclopentanone-2-carboxylate with bromoacetate, fol-
lowed by acidic hydrolysis and decarboxylation, afforded the
desired product 2k (Scheme 1).
Whole-cell mediated oxidations were carried out in shake
flasks as previously described (15a, 21). In most cases, one
equivalent of β-cyclodextrin (relative to the ketone) was in-
cluded in the reaction medium to diminish any of the toxic
effects of the substrates. The reactions were monitored by
chiral GC, and the concentrations of the ketone and the
lactone were measured against an internal standard (methyl
benzoate). Only the results that were consistent in terms of
conversion, enantioselectivity, time, and isolated yields over
the course of several fermentations are listed in Tables 1a
and 1b. Controlled chemical-oxidation experiments were
performed for all substrates, and it was possible to achieve
baseline resolution for the majority of ketones and (or) lac-
tones. The absolute configuration of ketones 2a and 2b were
deduced by comparison with literature values as described in
our earlier paper (20). The absolute configurations for 2c–f
were tentatively assigned by analogy with prior results (22)
© 2002 NRC Canada

Wang et al.
615
Scheme 2.
O
O
O
O
O
O
COOMe
COOMe
OH
OH
(CH2OH)2
LiAlH4
THF
87%
CeCl3.7H2O
NaI, RT, 30min
97%
p-TsOH,
RT, 30min
75%
4
3
2g
(1) NaH, R'X
89%
71%
O
O
O
O
90%
OR'
OAc
(2) H3O+,
acetone
OR'
2 j
2 h,i
4 h,i
and with the demonstrated selectivity of the CHMO for (S)-
cyclohexanones and cyclopentanones substituted with n-
alkyl chains (6, 23). It should be noted that because of
changes in numbering priority the R/S nomenclature is re-
versed for compounds 2a and 2c–k.
acted very slowly with yeast (CHMO) to give (R)-5g (34%
ee, [α]2D5 –11 (c 4.5, EtOAc)). The absolute configuration
for this compound was confirmed by comparison with the
previously reported rotation (25). The difficulty in transport-
ing the more polar substrates across cell membranes could
be partially responsible for the sluggishness of this transfor-
mation. When the hydroxy group in 2g was masked in the
form of its ether derivatives 2h and 2i, the oxidations were
faster, which supports the transport-through-membranes hy-
pothesis. Furthermore, the enantioselectivity of the latter
transformations was improved, particularly in the case of
ketone 2i bearing a long allyloxy chain. In this case, the
lactone product (R)-5i was obtained in 93% ee. When the
allyl group in lactone 5i was removed (Pd/C in p-
toluenesulfonic acid–H2O), the product 5g was shown to
have the same configuration (R) as the major lactone enan-
tiomer obtained from the oxidation of ketone 2g by yeast
(CHMO) or E. coli (CPMO). Again, the two enzymes
showed a preference for the same enantiomer, and again, the
CHMO enzyme proved to be the more selective of the two.
Ketone 2j was transformed by E. coli (CPMO) to the known
lactone (R)-5j (26). At 41% conversion and 53% ee the reac-
tion was stopped, the lactone product was isolated, and the
optical rotation was determined ([α]2D5 –15 (c 2.5, EtOAc)).
In a parallel reaction catalyzed by E. coli (CHMO), the 2j
substrate was also consumed and at 49% conversion an un-
identified optically active product was formed in 74% ee.
Lactone 5j from the CPMO oxidation was subsequently
chemically hydrolyzed (NaHCO3, MeOH, pH 11) to (R)-5g
without a change in optical purity. Ketone 2k was rapidly
oxidized by E. coli (CPMO), but the E-value was only 8. At
52% conversion, the product mixture consisted of lactone 5k
(61% ee, [α]2D5 –10 (c 1.2, EtOAc)) and residual ketone 2k
(66% ee, [α]2D5 –12.5 (c 0.8, EtOAc)). The transformation
with E. coli (CHMO) was very slow and apparently
nonselective and, since large quantities of by-products and
metabolites were formed in the early stages of the reaction,
we did not pursue this transformation any further.
A majority of the reactions reported in Tables 1a and 1b
were performed with the recombinant E. coli (CHMO); how-
ever, the same reactions that were re-investigated with the
yeast (CHMO) gave virtually identical results, apart from
small variations in the isolated yields. Compound 2d ap-
peared to be toxic to E. coli even in the presence of cyclo-
dextrin and the successful transformation could only be
achieved with the yeast (CHMO) strain. As can be seen in
Table 1a, branching and increased bulk of the 2-substituents
(2c–e) resulted in diminished selectivity of the CHMO oxi-
dations. Parallel E. coli (CPMO) catalyzed reactions were
generally faster and the lactones were isolated in excellent
yields; however, the enantioselectivity of the transformation
was consistently lower than that with CHMO. Extending the
three-carbon chain by a bromine atom in compound 2f re-
stored CHMO’s selectivity and the racemic ketone was
kinetically resolved to give the lactone 5f ([α]2D5 –63 (c 0.4,
EtOAc), 95% ee) and the unreacted ketone 2f ([α]2D5 –144
(c 1.65, CH2Cl2), 92% ee) in excellent isolated yields. The
(S)-2f ketone was subsequently chemically oxidized to the
corresponding lactone without loss of optical purity. Thus,
readily prepared racemic 2-(3′-bromopropyl)-cyclopentanone
(2f) introduces the possibility of further modification of the
highly enantiopure δ-valerolactones.
The E. coli (CPMO) catalyzed oxidations, shown in Ta-
ble 1a, were intentionally interrupted at low conversion lev-
els to estimate the enantioselectivity values (E) (24), which
turned out to be low, but consistently showed a slight prefer-
ence for the same ketone enantiomer as the CHMO-
catalyzed transformations. Uninterrupted E. coli (CPMO)
mediated reactions rapidly produced racemic lactones in ex-
cellent yields. All starting materials were generally con-
sumed, and neither by-products nor metabolites were evident
in the GC traces of the crude-reaction mixtures. Only in the
case of ketone 2f did the CPMO-mediated transformation
show significant enantioselectivity.
Discussion
The 2-cyclopentanones substituted with polar oxygen-
containing groups on the side chain were acceptable sub-
strates for both CHMO and CPMO (Table 1b). Ketone 2g,
with an unprotected hydroxy group on the side chain, re-
Cyclopentanones substituted at the 2-position with alkyl
chains of five carbons or longer are substrates to several
Baeyer-Villiger monooxygenases. Acinetobacter species
NCIB 9871 and TD 63 (23), Pseudomonas sp. NCIMB
© 2002 NRC Canada

616
Can. J. Chem. Vol. 80, 2002
Table 1a. Biological Baeyer-Villiger oxidations of 2-substituted cyclopentanones.
O
O
O
Baeyer-Villiger
biotransformation
R
R
+
O
R
2 (racemic)
2
5
Conv. %a Ketone 2
yield %b (ee %)c yield %b (ee %)c
Lactone 5
Ed
Organism
Time
(hours)
O
10 (51 S)
54 (34 R)
8
3
27
95
2.3
0
E. coli / CHMO
E. coli / CPMO
2a
95a (- -)
5a (- -)
O
32 (98 R)
89a ( 2 R)
18 (95 S)
11a (19 S)
12
50
11
200
1.5
E. coli / CHMO
E. coli / CPMO
2b
2c
2.5
O
O
O
28 (--)e
54a (--)e
20 ( 9)
46a (16)
33
24
37
46
1.3
1.6
E. coli / CHMO
E. coli / CPMO
18 (--)e
23 (83)
19
--
35
16
Yeast / CHMO
E. coli / CPMO
2d
2e
ND
28 (--)e
56 (58)
15a (26)
34
85
7.3
1.3
E. coli / CHMO 16
Ph
85a (--)e
16
E. coli / CPMO
O
47 (92)
64a (34)
47 (95)
36a (61)
49
36
128
5.7
Yeast / CHMO
E. coli / CPMO
24
36
Br
2f
a
b
Conversion from G.C.; Isolated yield of chromatographically purified product; c Determined from chiral G.C.(Supelco
β-Dex 225 column); d Calculated according to Ref.(24); e Not resolved on chiral GC; Note that because of change in
numbering priority the R/S nomenclature for ketones and and lactones a, c-f is reversed vis a vis that of 2b and 5b; also
absolute configurations indicated are only tentatively assigned for compounds c-f (see text).
10007, and enzyme fractions MO1 and MO2 from that or-
(10, 23, 27). With cyclopentanones, however, the chains
must be longer to obtain comparable enantioselectivities.
Cyclopentanones and cyclohexanones with functionalized
chains have not been evaluated as substrates for either
Acinetobacter sp. or the isolated cyclohexanone monooxy-
genase enzyme, but they were used as substrates in oxida-
tions with the MO2 fraction from the species NCIMB 10007
(7, 19) and NCIMB 9872 (7, 18, 19). These compounds
were of particular interest to us, since the oxidations with
our easy-to-use bioreagents could provide convenient routes
to optically pure lactones, ready for further modification
through classical chemical reactions.
Our earlier experiments (20) with the yeast (CHMO) have
shown that, although 2-n-butylcyclopentanone was fully
kinetically resolved over the course of the Baeyer-Villiger
oxidation, the 2-allylcyclopentanone was not, giving at best
the (R)-lactone with 51% ee. To investigate the effect of
branching and of polar substituents on the selectivity of
CHMO and CPMO, we evaluated several potential substrates
ganism were used in oxidations of these compounds. The
highest enantioselectivities were reported in reactions cata-
lyzed by the MO2 fraction (7). Acinetobacter-catalyzed re-
actions were found to be highly enantioselective in several
of the compounds studied, but the yields were low because
the lactonic products were further metabolized by a
hydrolase present in the organism. The problem was mini-
mized by adding tetraethyl pyrophosphate to the reaction
medium to inhibit the hydrolytic enzyme, and the reactions
were re-investigated using Acinetobacter TD 63, a related
species, which does not produce lactone hydrolase (23).
Using our nonpathogenic yeast (CHMO) strain, we were
able to achieve excellent kinetic resolution of the 2-alkyl-
cyclopentanones, provided that the chains were at least four-
carbon atoms long (20). It was previously observed that
cyclohexanone monooxygenase exhibits virtually complete
enantioselectivity for the (S)-enantiomer of 2-alkyl cyclo-
hexanones, with the exception of 2-methyl cyclohexanone
© 2002 NRC Canada

Wang et al.
617
Table 1b. Biological Baeyer-Villiger oxidations of 2-substituted cyclopentanones.
O
O
O
Baeyer-Villiger
biotransformation
R
R
(R)
+
O
(S)
R
2 (racemic)
2
5
Conv. %a
Ketone 2
Lactone 5
Ed
Time
(hours)
Organism
yield %b (ee %)c yield %b (ee %)c
O
10 (34 R)
5a (43 R)
20 (11 S)
95a (12 S)
24
2.3
2.8
Yeast / CHMO 96
E. coli / CPMO
OH
2g
4
5
66 ( --)e
22a (-- )e
2.8
3.7
O
O
O
E. coli / CHMO 36
E. coli / CPMO
14
78
14 (44 )
78a (56)
O
2h
6
19 (-- S)e
70a (-- S)e
69
13
E. coli / CHMO 36
E. coli / CPMO 19
45
30
33 (93 R)
30a (81 R)
O
2i
--
--
--
--
--
5
E. coli / CHMO
E. coli / CPMO
OAc
2j
41a (53 R)
41
59a (-- S)e
4.6
O
O
--
--
--
--
--
E. coli / CHMO
E. coli / CPMO
OEt
2k
48a (66)
52a (61)
8
10
52
O
Ketone 6a,b
yield %b (ee %) yield %b (ee %)
Lactone 7a,b
purified MO2f
OAc
6
--
3.5
0.5
13 (75 S)
37 (68 R)
34 (83 R)
59 (42 S)
17
5
purified CPMOg
61
a
b
Conversion from G.C.; Isolated yield of chromatographically purified product; c Determined from chiral G.C.(Supelco
β-Dex 225 column); d Calculated according to Ref.(24); e Not resolved on GC; f Ref. 23; g Ref. 7, 19. Note that because of
change in numbering priority the R/S nomenclature for ketones and lactones g-k is reversed vis a vis that of compounds 2b and
5b.
listed in Table 1. The ability of CHMO to accommodate po-
lar chains was intimated by the fact that both isolated en-
zyme (28) and our recombinant organisms successfully
oxidized cyclohexanones with polar chains in the 3- and 4-
positions (29). To the best of our knowledge, however, these
compounds have not been tested as substrates for CPMO.
The point that needs to be stressed involves generaliza-
tions applied to the enantioselectivity trends of various
oxygenases. Although these can be useful when one chooses
an enzyme or an organism for a specific transformation,
wide speculation, resulting in nonconfirmed assignments of
absolute configuration, are unwarranted. We experienced
this when comparing oxidations by CHMO and CPMO with
those performed by MO2. In an earlier study, Adger et al.
(18) showed that monooxygenase MO2 from camphor-
grown NCIMB 10007 species converted racemic 2-(2′-
acetoxyethyl)cyclohexanone (6) to the corresponding R-(–)-
lactone 7a (40% conversion, 83% ee), while purified CPMO
from cyclopentanol-grown NCIMB 9872 transformed the
same substrate to its S-(+) antipode 7b (59% conversion,
42% ee), as shown in Table 1b. The rotations were reported
and the absolute configurations were unambiguously con-
firmed after the two lactones were converted to the (R)- and
(S)-lipoic acids. MO2 was also used to oxidize cyclo-
pentanones 2j and 2k to lactones 5j and 5k, respectively, in
better than 98% ee (7). The two lactones were reported as
having the R configuration. Unfortunately, neither rotations
nor references were specified. We were able to unambigu-
ously identify lactone 5j from the CPMO oxidation as the
(R) enantiomer (vide supra), but the absolute configuration
© 2002 NRC Canada

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Can. J. Chem. Vol. 80, 2002
of lactone 5k has not yet been confirmed. Although it was
shown (19) that enantioselectivity of CPMO (NCIMB 9872)
can be opposite to that of MO2 (Table 1b, example 6), it ap-
pears that, at least for cyclopentanone 2j, the enantio-
selectivities are the same and that both enzymes
preferentially oxidize the (R) ketone.
dried over anhydrous potassium carbonate, distilled, and
stored over 3 Å molecular sieves. All solvents were purified
by fractional distillation. Other reagents were obtained from
commercial suppliers and used as received.
Protocols for biotransformations
In summary, the goals of this project were to determine
(i) if cyclopentanones with more polar functionalized sub-
stituents in the 2-position are substrates for CHMO and
CPMO; (ii) if highly optically enriched lactone (or lactones)
suitable for further chain modifications could be identified;
and (iii) if the performance of CPMO was adequately differ-
ent from the CHMO to attempt its development as an alter-
native bioreagent for biological Baeyer-Villiger reactions.
From the results reported here it is clear that while Baeyer-
Villiger oxidations of 2-substituted cyclopentanones by
CHMO- and CPMO-expressing strains are frequently
nonselective, some compounds are oxidized with a very high
enantiselectivity; in the present survey three of the candi-
dates investigated (2b, 2f, and 2i) were found to meet the se-
lectivity criteria. In general, of the two enzymes studied,
CHMO was shown to be the more selective. On the other
hand, oxidations with CPMO are appealing for another rea-
son: these transformations are fast, clean, and there are no
by-products or metabolites. Where enantioselectivity is not a
primary concern, the E. coli (CPMO) strain (16) can serve as
an excellent biological equivalent of organic peracids.
In conclusion, the results presented here indicate that “de-
signer microbes” expressing various Baeyer-Villiger mono-
oxygenases can contribute significantly to “developing
synthetic methodology and “green” processes that meet cri-
teria of a sustainable, environmentally conscious develop-
ment (30).”
Preparation of yeast cells (pKR001) and the general pro-
cedure for yeast-catalyzed Baeyer-Villiger oxidations were
previously described (14, 21, 22). Maintenance and
biotranformation protocols for the reactions with E. coli
[BL21(DE3)(pMM4)] are outlined in refs. 15 and 29.
Protocol for E. coli-(CPMO) mediated oxidations
The E. coli strain DH5α[pCMP201] was streaked from a
frozen stock on LB-ampicillin plates and incubated at 37°C
until colonies were 1–2 mm in size. One colony was used to
inoculate 10 mL of LB-ampicillin medium in a 50-mL
Erlenmeyer flask and was shaken at 30°C and 250 rpm over-
night. This culture was used at a 1:100 (v/v) ratio to inocu-
late an LB-ampicillin medium supplemented with 10%
glucose in a baffled-Erlenmeyer flask. The culture was incu-
bated at 30°C and 250 rpm until OD600 was approximately
1. IPTG (isopropyl thio-β-D-galactoside) stock solution
(200 mg per mL in water) was added (0.1 µL per mL of me-
dium) and the flask was shaken for another 30 min. The sub-
strate was then added, and, if solubility was a problem,
cyclodextrin was introduced at this stage. The culture was
agitated at 30°C at 250 rpm and monitored by GC or TLC.
The culture was saturated with NaCl and extracted with
ethyl acetate. Combined extracts were washed once with
brine and dried with anhydrous Na2SO4 or MgSO4. The sol-
vent was removed on a rotary evaporator and the residue was
purified by flash chromatography.
Compounds 2d and 2e
Experimental
Compounds 2d and 2e were prepared according to modi-
fied literature procedure (31). Anhydrous K2CO3 (5.5 g,
40 mmol) and acetone (20 mL) were placed in a three-neck
round-bottom flask equipped with reflux condenser. After
flushing the system with nitrogen, methyl cyclopentanone-2-
carboxylate (20 mmol) and alkyl halide (20 mmol) were
added, and the stirred reaction mixture, maintained under ni-
trogen, was gently heated at reflux for two days until all
starting material was consumed or a significant amount of
by-product was formed as shown by GC. The mixture was
cooled to room temperature and poured into ice water. The
aqueous phase was extracted with ethyl acetate (3 ×
200 mL) and the combined extracts were washed with brine
and dried over anhydrous Na2SO4. After removal of the sol-
vent on a rotary evaporator the crude product 1d (or 1e) was
purified by flash chromatography on silica gel using a mix-
ture of hexane and ethyl acetate as eluent. The resulting 2-
alkylketoester (1d or 1e, 10 mmol) was dissolved in a mix-
ture of glacial acetic acid (20 mL) and HCl (6 M, 10 mL)
and gently heated at reflux under nitrogen until all starting
material was consumed as shown by GC (usually 4–6 h).
The reaction mixture was cooled to room temperature,
poured into ice water, and extracted with ethyl acetate or pe-
troleum ether (3 or 4 × 150 mL). The combined extracts
were washed with water and then with saturated NaHCO3.
After drying over anhydrous Na2SO4, the solvent was
Optical rotations were measured on a PerkinElmer 241
polarimeter operating at room temperature. IR spectra were
recorded from thin films on a Mattson Satellite FT-IR spec-
1
trometer. H and 13C NMR spectra were recorded on Varian
XL-200 or Bruker AMX-400 FT-NMR spectrometers. All
spectra were recorded in CDCl3 solutions unless otherwise
specified. Chemical shifts are reported in ppm with TMS as
internal standard. EI-MS spectra and HRMS were obtained
on Kratos MS 50 mass spectrometer. Packed-column gas
chromatography was performed on a Shimadzu GC-9A gas
chromatograph employing a customer-packed column
(1/8" × 1 m, 5% OV-101 on 100/120 Supelcoport, Supelco
Inc.). Capillary gas chromatography was performed on a
Hewlett Packard 5890 instrument employing a 0.54 µm ×
1.00 mm × 15 m DB-1301 column (J&W) or Shimadzu GC-
9A using a 0.32 µm × 0.25 mm × 30 m β-Dex 225 column
(Supelco). All the GC instruments used flame-ionization de-
tectors and helium as the carrier gas. The injector and detec-
tor temperatures were maintained at 225°C and 300°C,
respectively. Thin-layer chromatography was performed on
precoated silica gel 60 plates (Aldrich). Flash chromatogra-
phy was performed on silica gel (200–425 mesh, Fisher Sci-
entific or SiliCycle). Tetrahydrofuran was distilled from Na
in the presence of benzophenone. Acetone was dried over
CaSO4 and distilled from KMnO4. Methylene chloride was
© 2002 NRC Canada

Wang et al.
619
removed and the crude product 2 was purified by vacuum
distillation.
2H), 1.52 (m, 2H). 13C NMR δ: 171.6, 136.4, 129.5, 128.5,
126.8, 81.0, 42.1, 29.4, 27.0, 18.4.
2-iso-Butyl cyclopentanone (2d)
(R)-6-(3′-Bromopropyl)tetrahydropyran-2-one (5f) (33)
Ketone 2f (100 mg, 0.45 mmol) and γ-cyclodextrin (0.5 g)
were added to YP-Gal (100 mL) in a 250-mL baffled-
conical flask. The mixture was then shaken at 30°C at
250 rpm for 5–10 min to obtain a uniform dispersion. A 1-
mL portion of yeast cells was added to the reaction flask and
the culture was shaken at 30°C (250 rpm). The reaction
monitored by GC and chiral-GC, and was stopped at 50%
conversion. After saturating the mixture with NaCl, the or-
ganic layer was extracted with ethyl acetate (3 × 100 mL).
The combined organic extracts were dried over anhydrous
sodium sulfate and concentrated by rotary evaporation. Sep-
aration of chiral lactone and ketone by flash chromatography
(hexane–acetone, 3:1) gave lactone 5f as colorless oil
(51 mg, 47% yield, 94% ee) and ketone (S)-2f also as color-
less oil (53 mg, 47% yield, 92% ee). (R)-5f: [α]2D5 –63
(c 0.4, EtOAc, 95% ee (GC)). IR νmax (neat): 2956, 1734 (s),
bp 120–140°C (18–25 Torr). IR νmax (neat) (cm–1): 2957.5
1
(s), 2869.1 (m), 1739.4 (s), 1467.1 (m), 1154.0 (m). H NMR
δ: 2.20 (m, 2H), 2.10 (m, 1H), 2.09–1.90 (m, 3H), 1.72 (m,
1H), 1.58 (m, 1H) 1.42 (m, 1H), 1.08, (m, 1H), 0.86 (d, J =
3.2 Hz, 3H), 0.82 (d, J = 3.0 Hz, 3H). 13C NMR δ: 221.8,
47.4, 38.9, 37.9, 30.0, 26.1, 23.3, 21.4, 20.7.
2-Benzylcyclopentanone (2e)
Obtained after chromatography (hexane–ethyl acetate
10:1) in 85% yield as pale yellow oil: IR υmax (neat) (cm–1):
3000 (m), 2960 (s), 2870 (m), 1730 (vs), 1600 (w), 1490
1
(m), 1450 (s), 1400 (m), 1150 (s), 690 (s). H NMR δ: 7.27
(m, 2H), 7.17 (m, 3H), 3.15 (dd, J = 7 and 3 Hz, 1H), 2.54
(dd, J = 7 and 6 Hz, 1H), 2.37 (m, 2H), 2.12 (m, 2H), 1.97
(m, 1H), 1.71 (m, 1H), 1.60 (m, 1H). 13C NMR δ: 220.1,
140.0, 128.8, 128.4, 126.1, 51.0, 38.2, 35.6, 29.1, 20.5.
1
1242, 1944. H NMR δ: 4.29 (m, 1H), 4.08 (m, 2H), 2.57
2-(3′-Bromopropyl)cyclopentanone (2f)
(m, 1H) 2.42 (m, 1H), 1.88–1.71 (dm, 6H), 1.51 (m, 2H).
13C NMR δ: 171.3, 80.0, 64.1, 32.5, 28.0, 25.1, 24.5, 18.7.
MS m/z: 223 ([M + 1]+), 141, 112, 99, 55. HRMS calcd. for
C8H13BrO2: 220.0099 / 222.0078; found: 220.0099 / 222.0079.
Unreacted ketone (42 mg, 42%) (S)-2f: [α]2D5 –144 (c 1.65,
CH2C12, 92% ee (GC)). An analytical sample of (S)-2d was
oxidized with m-chloproperbenzoic acid (TFA, CH2Cl2) to
give (S)-5f 92% ee (GC).
The racemic ketone (32) was purified by flash chromatog-
raphy on silica gel using 10:1 petroleum ether–acetone as
the eluant to give 2f as colourless oil (2.6 g, 41% yield after
two steps). IR νmax (neat) (cm –1): 2960 (m), 2875 (m), 1743
1
(s), 1374 (w), 1242 (m), 1157 (w). H NMR δ: 4.00 (t, J =
3 Hz, 2H), 2.23 (m, 2H), 2.03 (m, 1H), 1.80–1.60 (m, 4H),
1.46 (m, 2H), 1.27 (m, 2H). 13C NMR δ: 220.7, 64.1.3, 48.6,
38.0, 29.5, 26.6, 26.0, 20.6. MS m/z (%): 204/206 (1), 125
(100), 107 (38), 83 (26), 67 (37), 55 (85).
1,4-Dioxa-spiro[4.4]nonane-6-carboxylic acid methyl
ester (3)
Methyl cyclopentanone-2-carboxylate (5.0 mL, 40 mmol)
was added to a solution of p-toluene sulfonic acid (5.6 g,
40 mmol) in ethylene glycol (20 mL) and stirred at room
temperature for 30 min. The mixture was poured into 50 mL
1 M KOH solution saturated with NaCl and extracted with
ether (4 × 50 mL). The combined organic extracts were
washed with brine and dried over anhydrous Na2SO4. The
solvent was removed on a rotary evaporator to give 3 as a
colourless oil, 4.73 g (75%). IR νmax (neat) (cm–1): 2950 (s),
2890 (m), 1700 (vs), 1480 (m), 1350 (m), 1210 (s), 1040
(m). 1H NMR δ: 4.05–4.00 (m, 1H), 3.97–3.88 (m, 3H), 3.70
(s, 3H), 2.92 (t, J = 7.7 Hz, 1H), 2.12 (m, 1H), 1.98–1.79
(m, 4H), 1.67 (m, 1H). 13C NMR δ: 172.8, 118.3, 65.1, 64.5,
52.2, 51.7, 51.6, 36.7, 26.9, 22.0.
6-Cyclopentyltetrahydropyran-2-one (5c)
Ketone 2c (100 mg, 0.66 mmol) was oxidized using
E. coli (CHMO) and was purified by flash chromatography
on silica gel (petroleum ether–ethyl acetate, 5:1) to afford 5e
(22 mg, 20%, 9% ee). IR νmax (neat) (cm–1): 2952 (s), 2869
(m), 1731 (s), 1243 (m), 1176 (m). 1H NMR δ: 4.10, (m, 1H),
2.57 (m, 1Ha), 2.45 (m, 1Hb); 2.07 (m, 2H), 1.90 (m, 3H),
1.75–1.50 (m, 4H), 1.42 (m, 2H), 1.28 (m, 2H). 13C NMR δ:
172.3, 84.5, 45.2, 29.7, 28.9, 28.8, 27.1, 25.6, 25.5, 18.7.
6-iso-Butyltetrahydropyran-2-one (5d)
Biotransformation of 2d using E. coli (CHMO) followed
by flash chromatography (petroleum ether–acetone, 7:1 then
5:1) gave 5d (23%, 83% ee). IR νmax (neat) (cm–1): 2928 (s),
1
2855 (m), 1741 (s), 1463 (w), 1246 (m), 1047(m). H NMR
1,4-Dioxa-spiro[4.4]non-6-yl-methanol (4)
δ: 4.31 (m, 1H), 2.51 (m, 1H), 2.43 (m, 1H), 1.87 (m, 4H),
1.63 (m, 1H), 1.47 (m, 1H), 1.28 (m, 1H), 0.90 (d, J = 1 Hz,
3H), 0.88 (d, J = 1 Hz, 3H). 13C NMR δ: 172.0, 78.7, 44.9,
29.4, 28.3, 23.9, 23.0, 22.0, 18.4.
1,4-Dioxa-spiro[4.4]nonane-6-carboxylic acid methyl es-
ter (3, 3.8 g in 10 mL THF, 20 mmol) was added to a vigor-
ously stirred suspension of LiAlH4 (0.8 g, 21 mmol) in THF
(60 mL) at 0°C. The mixture was stirred for 1.5 h and al-
lowed to warm up to room temperature. The reaction was
quenched by pouring it into an ice cold saturated tartaric
acid solution and was then extracted with ethyl acetate (5 ×
40 mL). The combined organic extracts were dried over an-
hydrous Na2SO4 and the solvent was removed on a rotary
evaporator to give 4 as a colourless oil, 2.81 g (87%). IR
νmax (neat) (cm–1): 3500 (m, br), 2950 (s), 2890 (s), 2390 (s),
2280 (m), 1460 (m), 1390 (m), 1330 (m), 1160 (s), 1100
6-Benzyltetrahydropyran-2-one (5e)
Biotransformation of 2e (100 mg, 0.57 mmol) using
E. coli (CHMO) followed by flash chromatography (petro-
leum ether–ethyl acetate, 4:1) gave 5e pale yellow oil (28%,
61% ee). IR νmax (neat) (cm–1): 3060 (w), 3027 (w), 2951
(m), 2874 (m), 1733 (vs), 1496 (m), 1455 (m), 1238 (s),
1178 (m), 1040 (m), 749 (w), 700 (m). 1H NMR δ:
7.33–7.14 (m, 5H), 4.51 (m, 1H), 3.09–2.88 (d ABquartet,
J = 27 and 11 Hz, 2H), 2.57 (m, 1H), 2.44 (m, 1H), 1.89 (m,
1
(m), 1020 (s). H NMR δ: 3.96–3.84 (m, 4H), 3.66–3.54 (m,
© 2002 NRC Canada

620
Can. J. Chem. Vol. 80, 2002
2H), 2.64 (br s, 1H), 2.34–2.05 (m, 1H), 1.84–1.76 (m, 1H),
1.74–1.46 (m, 5H). 13C NMR δ: 119.0, 64.5, 64.0, 62.5,
47.0, 35.6, 25.6, 21.3.
less oil, 0.56 g (94%). IR νmax (neat) (cm–1): 2970 (s), 2920
1
(s), 2870 (s), 1730 (s), 1150 (m), 1120 (m). H NMR δ: 3.53
(m, 2H), 3.30 (s, 3H), 2.34–1.96 (m, 5H), 1.80 (m, 2H). 13C
NMR δ: 219.5, 71.5, 59.0, 49.4, 38.6, 27.1, 20.8.
2-Hydroxymethylcyclopentanone (2g) (34)
CeCl3·7H2O (1.86 g, 5 mmol) and KI (0.3 g, 1.8 mmol)
were mixed with 18 g silica gel (200–425 mesh) in acetone
(30 mL) and ground in a mortar. Compound 4 (0.96 g,
6 mmol) was added and the mixture was stirred at room
temperature for 30 min. The silica was filtered off and the
solid was washed with acetone. The solvent was removed on
a rotary evaporator and the residue was purified by flash
chromatography (hexane–ethyl acetate, 3:1) to give 2g as a
colourless oil, 0.67 g (97%). IR νmax (neat) (cm–1): 3427 (s,
br), 2966 (s), 2877 (s), 1734 (vs), 1404 (m), 1154 (m), 1054
6-Methoxymethyltetrahydropyran-2-one (5h)
Ketone 2h was oxidized using E. coli (CHMO) to give 5h,
which was purified by flash chromatography (petroleum
ether–acetone, 5:1, then petroleum ether–acetone, 3:1) to
give a colorless oil (16 mg, 14%, 44% ee). IR νmax (neat)
(cm–1): 2940 (m), 2881 (m), 2815 (w), 1736 (vs), 1249 (s),
1071 (s). 1H NMR δ: 4.46–3.90 (m, 1H), 3.55–3.47 (m, 1H),
3.36 (s, 3H), 3.28–3.18 (m, 1H), 2.62–2.53 (m, 1H), 2.43–2.34
(m, 1H), 1.98–1.87 (m, 2H), 1.74–1.62 (m, 2H). 13C NMR δ:
172.6, 79.0, 74.4, 59.4, 29.6, 24.5, 18.3. The unreacted 2h
was also isolated: 7% ee; [α]2D5 –17 (c 1.3, CH2Cl2).
1
(m). H NMR (acetone-d6) δ: 3.73–3.64 (m, 3H, including
an overlapping OH peak), 2.26–2.12 (m, 3H), 2.10–1.95 (m,
2H), 1.96–1.75 (m, 2H). 13C NMR (acetone-d6) δ: 219.5,
61.6, 51.7, 39.0, 27.1, 21.4.
6-Allyloxymethyl-1,4-dioxa-spiro[4.4]nonane (4i)
Compound 4i was synthesized according to the same pro-
tocol as 4h, except allyl bromide was used instead of methyl
iodide. The crude product was used in the following step
without purification.
6-Hydroxymethyltetrahydropyran-2-one ((R)-5g)
Ketone 2g oxidized using yeast (CHMO) was purified by
flash chromatography (petroleum ether–acetone, 2:1) to give
colorless oil (16 mg, 14%, 34% ee). [α]2D5 –11 (c 4.5, EtOAc.
Lit. (25) (R)-enantiomer: [α]2D5 –32 (c 1.3, CHC13)). IR νmax
(neat) (cm–1): 3394 (m, br), 2947 (m), 2881 (w), 1723 (vs),
2-Allyloxymethylcyclopentanone (2i)
Crude 4i dissolved in a mixture of acetone (20 mL) and
HCl (5 mL, 2 M) was stirred at room temperature for 2 h.
Acetone was removed by rotary evaporation and the residue
was extracted with CH2Cl2. Combined extracts were dried
over anhydrous Na2SO4 and the solvent was removed by ro-
tary evaporation. The crude product was purified by flash
chromatography (hexane–ethyl acetate, 6:1) to give 2j as a
colorless oil, 0.35 g (90% from 4i). IR νmax (neat): 3081 (w),
2967 (s), 2877 (s), 1737 (vs), 1647 (w), 1152 (s), 1088 (s),
1
1249 (s), 1058 (s). H NMR δ: 4.42–4.35 (m, 1H), 3.76 (dd,
J = 12.1 and 3.2 Hz, 1H), 3.66 (dd, J = 12.3 and 5.5 Hz,
1H), 2.64–2.54 (m, 1H), 2.48–2.39 (m, 1H), 2.00–1.90 (m,
1H), 1.90–1.81 (m, 2H), 1.81–1.64 (m, 2H). 13C NMR δ:
171.4, 81.0, 64.9, 29.6, 23.6, 18.3.
6-Methoxymethyl-1,4-dioxa-spiro[4.4]nonane (4h)
1
926 (s). H NMR δ: 5.84–5.73 (m, 1H), 5.20–5.04 (m, 2H),
NaH (2 g, 50% dispersion in mineral oil, excess) was
added to a 100-mL two-neck round-bottom flask and the
system was flushed with nitrogen. NaH was washed 3 times
with THF, and then was finally suspended in THF (10 mL).
MeI (2 mL, excess) was added, followed by 1,4-dioxa-
spiro[4.4]non-6-yl-methanol 4 (1.58 g in 40 mL THF,
10 mmol) with vigorous stirring. After reacting overnight,
the mixture was poured onto ice cubes and extracted with
ethyl acetate (3 × 35 mL). The combined extracts were
washed once with 0.2 M KOH, once briefly with 0.5 M HCl,
and once with brine. After drying over anhydrous K2CO3,
the solvent was removed on a rotary evaporator to give the
title product as a colourless oil, 1.11 g (89%). IR νmax (film)
(cm–1): 2950 (vs), 2870 (vs), 1450 (s), 1380 (s), 1320 (s),
1200 (s), 1100 (vs), 750 (s). The crude product was used in
the next step without further characterizations.
3.87 (dt, J = 5.5 and 1.4 Hz, 2H), 3.57–3.48 (m, 2H),
2.31–2.10 (m, 3H), 2.12–2.00 (m, 1H), 2.00–1.91 (m, 1H),
1.88–1.76 (m, 1H), 1.76–1.65 (m, 1H). 13C NMR δ: 219.3,
134.5, 116.6, 71.9, 68.9, 49.2, 38.5, 27.0, 20.7.
6-Allyloxymethyltetrahydropyran-2-one ((R)-5i)
Ketone 2i oxidized using E. coli (CHMO) was purified by
flash chromatography (petroleum ether–acetone 5:1 followed
by petroleum ether–acetone, 3:1). [α]2D5 = –14.1 (c 2.2, CDCl3).
IR νmax (neat) (cm–1): 3078 (w), 2947 (m), 2876 (m), 1726
1
(vs), 1453 (m), 1347 (m), 1240 (s), 1062 (s), 932 (m). H
NMR δ: 5.91–5.80 (m, 1H), 5.25 (dq, J = 17.3 and 1.5 Hz,
1H), 5.16 (qd, J = 10.4 and 1.2 Hz, 1H), 4.46–4.39 (m, 1H),
4.01 (dd, J = 5.5 and 1.5 Hz, 2H), 3.56 (m, 2H), 2.60–2.52
(m, 1H), 2.48–2.39 (m, 1H), 1.98–1.88 (m, 2H), 1.87–1.76
(m, 1H), 1.75–1.63 (m, 1H). 13C NMR δ: 171.2, 134.2,
117.3, 79.1, 72.5, 71.8, 29.6, 24.6, 18.2. MS m/z (%): 171
([M + 1]+, 0.2), 114 (48), 99 (58), 71 (100), 55 (28). The
isolated chiral ketone 2i, 19% yield, 81% ee: [α]2D5 – 56 (c
1.9, CH2Cl2). Lactone (R)-5i was hydrogenated (5% Pd/C in
methanol–water (2:1) containing 1% p-toluene sulfonic acid,
5 h) to give (R)-5g (60%, 93% ee) (25).
2-Methoxymethylcyclopentanone (2h)
6-Methoxymethyl-1,4-dioxa-spiro[4.4]nonane (4h, 0.8 g,
4.7 mmol) was dissolved in acetone (10 mL). In a separate
flask PdCl2 (30 mg) was dissolved in acetone (1 mL) and
acetonitrile (0.5 mL). The two solutions were combined and
TFA (1 mL) was added. The mixture was stirred at room
temperature for 3 h, poured into brine, and extracted with
ethyl acetate (3 × 35 mL). The combined extracts were dried
over anhydrous Na2SO4 and the solvent was removed on a
rotary evaporator. The residue was purified by flash chroma-
tography (hexane–ethyl acetate, 10:1) to give 2h as a colour-
Acknowledgments
Support by the Natural Sciences and Engineering Research
Council of Canada (NSERC) is gratefully acknowledged
© 2002 NRC Canada

Wang et al.
621
15. (a) G. Chen, M.M. Kayser, M.D. Mihovilovic, M.E. Mrstik,
C.A. Martinez, and J.D. Stewart. New J. Chem. 827 (1999);
(b) For the most recent CHMO expressions in E. coli., see.
S.D. Doig, L.M. O’Sullivan, S. Patel, J.M. Ward, and J.M.
Woodley. Enzyme Microb. Tech. 28, 265 (2001); (c) M.B.
Kneller, M.J. Cheesman, and A.E. Rettie. Biochem. Biophys.
Res. Commun. 282, 899 (2001).
(M.M.K.). The NMR spectra were recorded at the Atlantic
Regional Magnetic Resonance Centre at Dalhousie Univer-
sity, Halifax, Canada, and we would particularly like to ex-
press our thanks for the Centre’s help. The authors also
thank Cerestar Inc. for supplying the cyclodextrin used in
this study.
16. H. Iwaki, Y. Hasegawa, P.C.K. Lau, S. Wang, and M.M.
Kayser. Submitted.
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