Microbiological transformations 60. Enantioconvergent baeyer-villiger oxidation via a combined whole cells and ionic exchange resin-catalysed dynamic kinetic resolution process

Article abstract of DOI:10.1002/adsc.200505048

A dynamic kinetic resolution process was applied to an enantioconvergent microbial Baeyer-Villiger oxidation of benzyloxycyclopentanone, rac-1. This was achieved by combining a whole cell-based kinetic resolution and an anion exchange resin-catalysed in s

Full text of DOI:10.1002/adsc.200505048

FULL PAPERS
Microbiological Transformations 60. Enantioconvergent
Baeyer–Villiger Oxidation via a Combined Whole Cells and
Ionic Exchange Resin-Catalysed Dynamic Kinetic Resolution
Process
´
´
´
Maria-Concepcion Gutierrez, Roland Furstoss, Veronique Alphand*
´
´
´
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Groupe Biocatalyse et Chimie Fine, FRE CNRS 2712, Universite de la Mediterranee, Faculte des Sciences de Luminy,
Case 901, 163 avenue de Luminy, 13288 Marseille Cedex 9, France
Fax: þ33(0)4-91-82-91-45, e-mail: alphand@luminy.univ-mrs.fr
Received: January 25, 2005; Accepted: April 1, 2005
Supporting Information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: A dynamic kinetic resolution process was ment of usable substrate concentration and afforded
applied to an enantioconvergent microbial Baeyer– nearly enantiopure lactone (R)-2 (97% ee) in prepa-
Villiger oxidation of benzyloxycyclopentanone, rac- rative (isolated) yields higher than 84%. Noyoriꢀs
1. This was achieved by combining a whole cell-based model was applied to this reaction and allowed kinet-
kinetic resolution and an anion exchange resin-cata- ic constants determination.
lysed in situ racemisation. Several resins were
screened according to their capacity to catalyse the
racemisation of 1. As compared to our previous re- Keywords: asymmetric Baeyer–Villiger oxidation; bio-
sults, the presence of weakly basic Lewatit MP62 in transformations; dynamic kinetic resolution; ion ex-
biotransformations of 1 allowed a three-fold improve- change resin; racemisation; whole-cell process
Introduction
The Baeyer–Villiger oxidation of ketones into lac-
tones is an important reaction in organic chemistry be-
The increasing interest and need for enantiomerically cause of its potential applications in synthesis, lactones
pure compounds in pharmaceutical and agricultural in- being valuable building blocks for synthesis of countless
dustry has led to considerable development of asymmet- natural – or unnatural – biologically active products.
ric catalysis techniques during the last decades.[1] In this Only recently, transition metal-based chemical ap-
context, significant effort has been made to overcome proaches to asymmetric BV oxidation have been devel-
the important productivity limitation linked to the use oped with, to date, generally rather moderate success.[7,8]
of classical resolution processes – i.e., the maximum On the other hand, we (and others) have demonstrated
50% theoretical product yield barrier – and new strat- that specific enzymes – i.e., Baeyer–Villiger monooxy-
egies have been developed to afford the enantiopure genases[9] (BVMOs) – were highly efficient to achieve
product in nearly quantitative yields. One possible way asymmetric BVoxidation with high enantio- or enantio-
to reach this goal is dynamic kinetic resolution toposelectivity.[10,11] Moreover, we recently have devel-
(DKR),[2] an approach which combines an in situ race- oped a logistically simple whole cell process involving
misation with a kinetic resolution (KR) step (cf. a resin-based in situ SFPR (substrate feeding and prod-
Scheme 1). Both substrate enantiomers can thus be con- uct removal) strategy, which allowed us to overcome one
verted into a single enantiomer of the product with a of the main bottlenecks[11] met with this type of biotrans-
100% theoretical yield. A number of successful exam- formation, i.e., the low substrate or product concentra-
ples for DKR methods have been reviewed recently.[3] tion dictated by inhibition phenomena. The successful
Some of them involve solely conventional chemical application of such a process to the biotransformation
methods,[4] whereas others associate chemical and bio- of rac-bicyclo[3.2.0]hept-2-en-6-one has opened the
catalytic steps.[5] In this latter case, enzymes or whole way to highly productive biooxidation.[12]
cells contribute either to racemisation[6] or to kinetic res-
olution.[3]
In the same context, we also have recently reported a
first example of DKR.[13] This was applied to 2-benzyl-
oxymethyl-cyclopentanone, rac-1 (Scheme 1), and was
Adv. Synth. Catal. 2005, 347, 1051–1059
DOI: 10.1002/adsc.200505048
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both these compounds were isolated in, respectively,
54 and 33% yield and 84 and 82% ee, which corresponds
to an apparent E value of ca. 30. On the other hand, the
DKR of rac-1 (300 mg, 1.5 mmol) carried out at pH 8.5
(using a 1-L cell broth) afforded (R)-2 (275 mg, 87%
yield) in 96% ee.[13]
However, drawbacks in this interesting DKR process
were (a) limited ketone concentration (0.3 g Lꢀ1), owing
to potential toxicity or inhibition effects of rac-1 towards
the microbial cells and/or the enzyme[18] and (b) signifi-
cant (35–40%) loss of enzymatic activity at the rather
high pH (8.5) yet essential for a fast enough racemisa-
tion of 1 (cf. Figure 1). As a consequence, a stringent
condition for improving this process would be to con-
duct the biotransformation at nearly neutral pH (which
Scheme 1. DKR of rac-1.
based on the combined use of a recombinant strain over- we have shown to be optimum as far as cell activity is
expressing CHMO (the best known BVMO)[14] and of a concerned – cf. Figure 1), while still performing fast
base-catalysed substrate racemisation (at pH 8.5). How- enough in situ racemisation. Moreover, since the rate ra-
ever, the partial loss of enzymatic activity observed at tio between the biooxidation on one side and the racemi-
this pH, as well as the rather low optimal usable sub- sation on the other determines the efficiency of a DKR
strate concentration, seriously hampered the prepara- procedure, the enzymatic activity enhancement allowed
tive scale applicability of this approach. We therefore by the use of an optimum (neutral) pH necessitated an –
have furtherinvestigatednewalternativesfor improving at least similar – improvement in the racemisation rate.
such a DKR process and, in the present work, we de-
scribe results based on the combined use of the whole-
cells approach and substrate racemisation catalysed, at Racemisation Studies
nearly neutral pH, by an ionic exchange resin.
To the best of our knowledge, only few studies have de-
scribed accurate racemisation kinetics.[19,20] Zwanen-
Results and Discussion
burg and co-workers have recently summarised and
classified, in an excellent review, different racemisation
As already underlined by Faber,[2] the success of a DKR techniques.[21] The most common method is a base-cata-
process requires that the rate constant for racemisation lysed process generally performed at rather high pH,
(krac) is equal to (or greater than) the rate constant for which necessitates that the substrate bears an acidic hy-
transforming the substrate into product (kfast). Obvious- drogen at the stereogenic centre. However, a few exam-
ly, the compatibility of both reactions (racemisation and plesusinganionicexchangeresinshavealsobeendescrib-
enantioselective transformation) is the critical facet of
such a methodology, since substrate racemising condi-
tions must not adversely affect the resolution reaction,
or interfere with the chiral integrity of the product.
Thus, the main challenge in our study was to find sub-
strate-racemising conditions while not affecting the effi-
ciency of the enzymatic resolution.
As a model substrate for a-substituted cyclopenta-
nones, 2-benzyloxymethylcyclopentanone, rac-1, was
synthesised from commercial ethyl 2-oxocyclopentane-
carboxylate, following described experimental proce-
dures (four steps, 50% overall yield).[15] We had previ-
ously reported that microbial BVoxidation of rac-1, car-
ried out at nearly neutral pH, was very efficient using ei-
ther a culture of the wild-type bacteria Acinetobacter
calcoaceticus NCIMB 9871[13] or a culture of the re-
combinant E. coli TOP10[pQR239] strain overexpress-
ing CHMO.[16,17] Thus, the (R)-1 enantiomer gave prefer-
entially nearly enantiomerically pure (R)-6-benzyloxy-
methyltetrahydro-2-pyrone 2, whereas (S)-1 was insig-
Figure 1. Relative specific activity of E. coli [pQR239] cells at
various pH values (measured against racemic bicyclo[3.2.0]-
nificantly oxidised. Using E. coli TOP10[pQR239], hept-2-en-6-one).
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Table 1. Characteristics of anion exchange resins and effect on the racemisation of 1.
Anion exchange resin
Strength and type of resin
Functional group
Exchange Measured Racemi-
capacity
pH[a]
sation
accelera-
tion
[meq gꢀ1
]
Amberlite IRA 900
Dianion HPA 25
Dowex Marathon MSA Strong (type I), macroporous
Lewatit OC1950
Amberlite IRA 910
Lewatit MP64
Strong (type I), macroreticular quaternary amine
3.5
2.2
4.1
1.1
3.5
6.9
6.9
7.0
7.4
7.3
7.8
7.9
7.7
–
–
–
–
Strong (type I), highly porous
quaternary alkyl amine
trimethylbenzylammonium
quaternary amine
Strong (type I), gel
Strong (type II), macroreticular dimethylethanolamine
–
Medium, macroporous
Weak, macroporous
Weak, gel
Tertiary and quaternary amine 1.4
Tertiary amine
polyamine
þ þ
þ þ þ
þ
Lewatit MP62
Amberlite IRA 67
1.7
4.9
[a]
pH measured after resin addition (12.5 meq Lꢀ1) into a culture medium at initial pH 6.7.
ed.[22,23] We therefore hypothesised that racemisation
Preliminary Controls
of the structurally simple a-substituted ketone rac-1
could, similarly, be performed at nearly neutral pH via As a consequence of the above observations and propos-
a ketone/enol equilibrium using such a resin. In order al for optimal experimental conditions, it remained to
to check this possibility, we first prepared enantiomeri- verify the compatibility of these conditions with the
cally enriched (S)-1 (65% ee) by performing the E. preservation of CHMO enzymatic activity and deter-
coli TOP10[pQR239]-catalysed biotransformation of mine the optimum usable substrate concentration. The
0.5 g of rac-1 at neutral pH. Using this enantiomerically CHMO activity of the recombinant E. coli strain was
enriched substrate, several strongly, medium and weakly checked by using racemic bicyclo[3.2.0]hepten-2-one
basic anion exchanger resins (gel and macroporous as a test substrate.[12] We observed that: (a) the anion ex-
types listed on Table 1) were screened for their racemi- changer Lewatit MP62 (12.5 meq Lꢀ1) only scarcely
sation performances.
affected the CHMO activity and (b) at pH 7, the temper-
Racemisation assays were carried out in the culture ature rise from 30 to 378C approximately led to a two-
medium at two different temperatures (30 and 378C) fold enzymatic activity increase (results not shown).
in the presence of 5 equivalents of resin with respect to We therefore proceeded to the evaluation of the DKR
the substrate. Racemisation is supposed to be a pseu- of rac-1 using these experimental conditions.
do-first-order reaction and rate constants (krac) were de-
In order to define the optimal ketone concentration
termined by a least square fit of the calculated and meas- that could be used, substrate and product inhibitory lev-
ured ee of 1 according to Equation (1):
els had also to be determined. The solubility of rac-1 in
ð1Þ
The results are reported in Figure 2. Because addition of
resin slightly modified the pH of the medium (cf. Ta-
ble 1) and in order to avoid a subsequent bias of out-
comes, the rate constant was compared with those of
the pH-dependent racemisation (black lines) at the con-
sidered pH. Thus, we observed that with type I and type
II strong anionic resins, which are the most frequently
used in experiments involving a heterogeneous base-
catalysed racemisation,[22, 23] the racemisation rates are
similar to those observed in the culture medium at the
corresponding pHs. A weak anion exchanger (IRA 67)
only led to modest improvement whereas the best re-
sults were obtained with the medium anionic resin Lew-
atit MP64 as well as with the weakly anionic resin Lew-
atit MP62. Interestingly, both these resins are macropo-
rous and are the only ones to bear tertiary amines.[24]
Lewatit MP62 was selected for further studies because,
in the presence of this resin, racemisation of 1 at 378C
(pH 7.7) was faster than at pH 9 at 308C (cf. Figure 2).
Figure 2. Racemisation constants (krac) with respect to pH.
Empty symbols: racemisation at 308C, filled symbols: racemi-
sation at 378C; full lines, black circle and empty circle: pH-de-
pendent racemisation (addition of KOH to the culture me-
dium); other single symbols: racemisation with various anion-
ic resins (12.5 meq Lꢀ1),
and
Lewatit MP62,
and
^
^
~
~
&
&
Lewatit MP64, and other resins.
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the culture medium was determined to be about 1.3 g Lꢀ broth before extraction and use of aDean–Stark appara-
1. Thus, several experiments achieved at different con- tus was necessary to ensure the cyclisation of hydroxy
centrations (pH 7, 308C, 250 mL shake flasks – results acid into lactone.
not reported) showed that rac-1 was not inhibitory until
a concentration close to its solubility (no decrease of the
reaction rates). Nevertheless, biooxidation did not go to Analytical Scale Experiments
completion at concentrations higher than 1 g Lꢀ1
.
Experiments were similarly carried out after prior ad- In order to check the influence of the resin on the bio-
dition of lactone rac-2 (synthesised by chemical BV ox- transformation of rac-1, we carried out experiments ei-
idation of 1) into the biotransformation medium (pH 7), ther in absence or in presence of Lewatit MP62 and, in
at various concentrations (cf. Figure 3). The initialꢀp1res- the latter case, the resin was added either at the begin-
ence of lactone 2 at a concentration of about 1 g L no- ning of the biotransformation or after 3.5 h. These ex-
ticeably slowed down the reaction rate. At 1.5 g Lꢀ1 ini- periments were conducted on an analytical scale in an
tial lactone concentration, biotransformation was total- aerated bottle at 1 g Lꢀ1 of rac-1. The concentration
ly inhibited. Consequently, it appeared that the optimal and ee of the unreacted ketone 1 are reported on Fig-
concentration of rac-1 usable for this reaction was close ure 4. In the absence of resin, the ketone ee raised up
to 1 g Lꢀ1. It is to be stressed that the observed inhibition to 70% before stabilising but, as soon as the resin was
might be due either to the cyclic (lactone) or to the open added, it decreased rapidly down to about 15%, a value
(hydroxy acid) form of 2, since both exist in equilibrium similar to the one reached when the resin was added at
in these experimental conditions.[25] In contrast, at the start of the bioconversion. We observed also a faster
pH 8.5, only the hydroxy-carboxylate would exist that disappearance of the ketone in presence of the resin. Af-
might explain the lower usable concentration of 1 ter extraction in recyclisation conditions, (R)-2 was ob-
(0.3 g Lꢀ1) at this pH,[13] suggesting that the open anionic tained with a yield as high as 83% and excellent ee
form would be more inhibitory than the protonated one. (>98%). These experiments unambiguously illustrate
the interest of this resin which makes possible perform-
ing this DKR of rac-1 at nearly neutral pH.
Dynamic Kinetic Resolution of rac-1
Studies on the BV biooxidation of rac-1 were carried out Further Improvements
at analytical and preparative scales, using a cell broth
regulated at pH 7.5. However, due to the above cited In order to improve this process, we further explored the
equilibrium between the open and closed form of 2, possible influence of reaction temperature and the pro-
and, to some extent, to a partial adsorption of 2 onto portion of resin used. As far as temperature is con-
the resin, accurate measurement of the lactone concen- cerned, we had previously observed that the BVase ac-
tration was not possible during the biotransformation. tivity of our E. coli recombinant strain (against bicyclo-
For preparative scale experiments, acidification of cell heptenone) was nearly doubled at 378C as compared to
Figure 3. Determination of the inhibitory level of lactone 2. Time dependent concentration of rac-1 in the presence of various
ꢀ1
initial concentrations of rac-2: 0, 0.5, 0.7, 1.0 and 1.5 g L (other conditions: 308C, pH 7, 0.5 g Lꢀ1 of rac-1).
^
~
~
*
*
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Enantioconvergent Baeyer–Villiger Oxidation
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*
Figure 4. Analytical scale DKR of rac-1. Effect of Lewatit MP62ꢀ1on unreacted ketone ees. No resin: ; addition of resin
(24.5 meq Lꢀ1) at time 0: ; after 3.5 h: (other conditions: 1 g L of rac-1, pH 7.5, 308C).
~
^
Kinetic Calculations
308C. As the temperature enhancement also increased
the racemisation rate, that could be an interesting im-
provement factor, provided that the increase of the rac- Interestingly, these observed drops of ee could be ex-
emisation rate would be at least equivalent to that of the plained using a theoretical kinetic model previously de-
enzymatic activity. Therefore, we carried out a series of veloped by Noyori and co-workers.[26] It was based on
analytical experiments at 378C (a) in the absence of res- two main hypotheses: (a) the reaction rates (krac, kR
in and (b) in the presence of 2.5 and 5 equivalents of and kS) of such a DKR process are pseudo-first order
Lewatit MP62 (corresponding, respectively, to 4 and in substrate concentration and (b) the kinetic resolution
8 meq Lꢀ1). Results are reported in Figure 5 and Ta- is an irreversible reaction. Assuming these hypotheses
ble 2. They indicate that the “stabilised” ketone ee are fulfilled in our experiments, we calculated the kinet-
reached a value higher than in the previous experiments ic constants of these reactions using a least squares fit
at 308C. Moreover, the ee of the formed lactone de- based on our experimentally determined ketone and
creases until a value of 68% after 6 h of bioconversion lactone ees. The results obtained are indicated in Ta-
when the reaction was conducted in the absence of resin, ble 2.
whereas values of 83 and 86% were observed in the
The good correlation we observed between the calcu-
presence of, respectively, 2.5 and 5 equivalents of Lewa- lated krac values and those determined independently in
tit MP62 (cf. Table 2).
the course of base-catalysed racemisation experiments
(cf. Table 2) as well as between calculated and experi-
mental values (black lines and symbols – cf. Figure 5),
seems to validate, in a first approximation, the applica-
bility of Noyoriꢀs kinetic model to our case. Calculations
Table 2. Kinetic constants determined from experimental ketone and lactone ees following Noyoriꢀs equations.
Resin concentration [meq mLꢀ1
]
0
4
8
Observed maximum ketone ee [%]
88
68
70
83
0.2
1.1
0.03
60
86
Observed lactone ee after 6 h reaction [%]
Calculated krac [hꢀ1
]
0.08 (0.08[a]
)
0.4 (0.5[a]
)
Calculated kR [hꢀ1
]
1.4
0.05
1.3
0.04
Calculated kS [hꢀ1
]
[a]
krac values determined separately during racemisation experiments.
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Figure 5. Analytical scale DKR of rac-1: Experimental and simulated effect of Lewatit MP62 on ketone and lactone ees. Ex-
*
*
perimental (symbol) and simulated (ꢀli1ne) ketone (empty symbol) and lactone (black symbol) ees. No resin: and ; Lewatit
ꢀ1
MP62 4 meq Lꢀ1
:
and ; 8 meq L
:
and (others conditions: 0.3 g L of 1, pH 7.5, 378C).
&
&
~
~
led to nearly unchanged kR and kS values in the presence scale experiments were performed, using different ex-
or absence of resin, whereas a noticeable increase of krac perimental conditions.
with resin concentration was observed – which translat-
A first run was carried out using 1 g of rac-1
ed into an increase of the ee of the formed lactone. These (4.9 mmoles), at 308C and in the presence of 5 equiva-
outcomes illustrate the necessity of a fast enough race- lents of Lewatit MP62. The two others were conducted
misation rate for achieving a successful DKR process. on, respectively, 1 g and 1.3 g of rac-1, at 378C and using
Thus, with an observed kR/kS ratio of ca. 30, krac has to 2.5 equivalents only of resin. In all cases, the pH was au-
be equal to, at least, one third of the kR value in order tomatically set at 7.5 until completion of the reaction.
to provide a lactone ee higher than 90%. Also, it is note- The results are reported in Table 3. First of all, in all
worthy that a two-fold increase of the resin proportion three cases and whatever resin proportion, a very satis-
only had a very negligible – if any – effect on the increase factory yield (higher than 84%) of purified lactone
of the lactone ee (86 versus 83%).
(R)-2 was obtained, indicating that the biooxidation
smoothly occurred indeed and that an efficient extrac-
tion of the lactone could be achieved. Further informa-
tion can also be drawn from these experiments: (a) a no-
ticeable acceleration of the biooxidation rate was ob-
Preparative-Scale Experiments
In order to validate the above determined optimal con- served at 378C. Thus, in these experiments (entry 2
ditions, and also to check that the formed lactone was and 3), 0.6 g Lꢀ1 of 2 were formed after 5 h biotransfor-
not irreversibly adsorbed onto the resin, three gram- mation, as compared to about 0.35 g Lꢀ1 in the 308C run
Table 3. Preparative DKRs of rac-1.
Entry Temperature Initial ketone Lewatit MP62
Residual ketone 1
Lactone 2
[8C]
concentration
Resin
concentration
yield[a]
ee max.
[%]
yield[a]
[%] (mass [g])
ee [%]
[g Lꢀ1
]
equivalents [meq Lꢀ1] (mass [g]) [%]
1
2
3
30
37
37
1
1
1.3
5
2.5
2.5
24 (14.6)
12 (7.3)
16 (9.5)
7
5
17
17
29
17
84 (0.90)
85 (0.93)
89[b] (1.04)
97
91
96
[a]
[b]
Isolated yield.
Yield based on the recovered ketone.
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Enantioconvergent Baeyer–Villiger Oxidation
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then with water until neutrality and filtrated under vacuum.
The number of resin equivalents (neq) used in this paper and
the resin concentration (cresin) were defined as follows, Equa-
tion (2) and Equation (3):
(entry 1), (b) however, for the 378C run (entry 2), the fi-
nal ee of the formed lactone 2 was lower whereas the ee
of the remaining ketone stayed higher throughout the
biotransformation, indicating that the racemisation
rate was not fast enough in these experimental condi-
tions, (c) interestingly, increasing the resin concentra-
tion (entry 3) had, as a result, a better ee (96%) of the
lactone, (d) on the other hand, a higher substrate con-
centration (1.3 g Lꢀ1) severely slowed down the biooxi-
dation rate after 10 h (17% of ketone still remained in
the medium), confirming the inhibitory effect of the lac-
ð2Þ
and
ð3Þ
tone at a concentration of ca. 1 g Lꢀ1
.
m being the mass of resin (g), n the ketone amount (mmoles), V
the volume (L) and C the resin exchange capacity expressed in
milli-equivalents per gram of resin (meq gꢀ1).
Conclusion
We have shown in this study that a very satisfactory dy-
namic kinetic resolution of ketone rac-1 could be set up
by combining whole-cell catalysed Baeyer–Villiger oxi-
dation (using a culture of a recombinant E. coli strain
overexpressing cyclohexanone monooxygenase from
Acinetobacter calcoaceticus) and in situ racemisation
catalysed by the weakly basic anion exchanger Lewatit
MP62. The choice of the adequate resin was the key
point of this work. Also, optimisation of the experimen-
tal conditions allowed us to define the parameters lead-
ing to both a high enzymatic activity and a fast enough
racemisation rate of the unreacted substrate. Noyoriꢀs
model was applied to this DKR in an attempt of ration-
alisation and allowed kinetic constants determination.
As compared to our previous results conducted at
pH 8.5, a three-fold substrate concentration could be
used, which translated into an equivalent increase of
productivity of this process. Starting from rac-1, nearly
enantiopure lactone (R)-2 (97% ee) was obtained in a
preparative (isolated) yield higher than 84%. Further
studies are in progress in our laboratory to improve
this type of DKR methodology.
Preparation of the Biocatalyst
The strain E. coli TOP10[pQR 239] contains a pBAD plasmid
into which the CHMO gene from A. calcoaceticus NCIMB
9871 has been cloned. The expression of the CHMO gene
was induced by l(þ)-arabinose. The culture medium contained
10 g Lꢀ1 each of glycerol, yeast extract (Fischer Scientific), soy-
bean peptone (Fluka), NaCl, as well as 100 mg Lꢀ1 of ampicil-
lin. An 11-L fermentor was filled with 5 L of culture medium
and was inoculated with 450 mL of a 12 h preculture at 378C.
The fermentor was stirred at 500 rpm and set at 378C, and
the vessel was aerated with an air flow of 1.3 vvm. The pH
was adjusted at 7.0 and controlled by automatic addition of
1 N H3PO4 and 1 N KOH solutions. l(þ)-arabinose (0.05%
w/v) was added to induce the enzymatic activity during the ex-
ponential growth phase (typically, OD590 nm ¼8). After a fur-
ther 1–2 h growth period, the cells were harvested and kept
at 48C overnight. They were directly utilised for biotransfor-
mation.
Analytics
Broth aliquots (3–5 mL) were extracted with ethyl acetate
containing octadecane (0.5 g Lꢀ1) as internal standard. The or-
ganic phase was then analysed by GC on a Optima-5 column
(Macherey-Nagel) at 2308C to determine the ketone 1 concen-
tration. The ee values of 1 (respectively, lactone 2) were deter-
mined using a Chirasil-Dex CB column (Chrompack) at 1508C
[respectively, a LipodexꢂE column (Macherey-Nagel) at
1708C].
Experimental Section
General Remarks
Gas chromatography analyses were performed using a Shi-
madzu GC-14A chromatograph with helium as the carrier gas.
E. coli TOP10[pQR239] was constructed by Prof. J. Ward
(University College, London). Stock cultures were grown on
nutrient agar at 378C and stored at ꢀ208C in a 40% glycerol
solution. Growth of microorganisms was carried out in an 11-
L (New Brunswick) fermentor equipped with a DOT probe,
pH control and antifoam automatic addition. Analytical scale
biotransformation experiments were carried out in a specially
built 500-mL glass bubble column equipped with a sintered
glass at the bottom[12a, c] and preparative experiments in a 2-L
(Setric) fermentor.
Absolute configurations of ketone 1 and lactone 2 were de-
duced by comparison with GC retention times of authentic
samples as described in ref.[13]
Racemisation Studies
Racemisation experiments with optically active cyclopenta-
none 1 (isolated from the preparative kinetic resolution per-
formed at pH 7 using same the E. coli strain) were performed
into 2-mL Eppendorf vials, shaken on a reciprocal shaker at
the appropriate temperature (30 or 378C). Ketone 1 (predis-
Anion exchanger resins were purchase from Supelco or Flu-
ka. Before use, they were washed with NaOH solution (3 N),
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Maria-Concepcion Gutierrez et al.
FULL PAPERS
solved in an EtOH solution) was added to obtain a final con-
centration of 0.5 g Lꢀ1. Racemisation studies versus pH were
performed using the culture medium adjusted to the selected
pH value by addition of a KOH solution. Racemisation studies
with ionic exchange resins were carried out in the culture me-
dium after addition of 5 equivalents of resin per moles of 1 – i.e.,
ata concentrationof 12.5 meq Lꢀ1. Ketone ees were monitored
by chiral GC analysis of samplings (80 mL) kept at þ48C after
AcOEt extraction. Results are summarised in Table 1 and Fig-
ure 2.
Calculations: Kinetic constants (krac, kR, kS) were deter-
mined by a least squares fit from Noyoriꢀs equations[26] using
ketone and lactone ees measured as described above during
the DKR (Scientis software).
Preparative Scale Biotransformations
Typically, preparative scale biotransformations of cyclopenta-
none 1 (dissolved in 10 mL EtOH) were carried out in a 2-L fer-
mentor filled with 1 L of a E. coli TOP10[pQR239] cells. Agi-
tation was fixed at 500 rpm and the air flow at 16 L hꢀ1. The ap-
propriate pH value (pH 7.5) was adjusted and controlled by au-
tomatic addition of KOH (3 N) or H3PO4 solution (l N). Bioox-
idation was monitored by GC analysis of aliquots and stopped
at completion by addition of 1 N H2SO4 until pH 6. The me-
dium was then continuously extracted with dichloromethane
24 h before being acidified down to pH 2 and extracted again
for 24 h. Crude extracts were pooled and refluxed in anhydrous
toluene (using a Dean–Stark apparatus) containing Amberlite
IR 120 resin before immediate purification by flash chroma-
tography (pentane/AcOEt).
Activity Measurement
Whole cell CHMO activity was measured against commercial
(ꢁ)-bicyclo[3.2.0]hept-2-en-6-one (0.5 g Lꢀ1) using 100 mL
culture broth in a 500-mL bubble column reactor. Air was
sparged through a sintered glass bottom at a flow rate of
1.3 vvm and further agitation was not necessary to ensure
good mixing. A water jacket maintained the temperature at
the chosen value. pH was controlled by automatic addition of
1 N H3PO4 and 1 N KOH solutions. The experiments were car-
ried out at different pH values, i. e., 6.8, 7.5, 8 and 9, and at two
different temperatures: 30 and 378C. One assay was performed
by addition of 1.45 g (2.5 resin equivs.) of Lewatit MP62 at
pH 7.5 and 308C. Aliquots were taken at regular time intervals,
extracted with ethyl acetate containing undecane as internal
standard and analysed by GC (Optima 5 column, 1108C). Spe-
cific whole-cell activity was calculated in U/g cells (dry weight)
from the initial velocity of combined lactone formation.
Acknowledgements
This work was partly funded by the Project QLK3-CT2001-
00403 “BIOMEX” as part of the European Community Fifth
Framework Programme. We would like to express our gratitude
to Prof. J. Ward, for having provided us with the recombinant
bacterial strain. M. C. G. would like to express her best acknowl-
´
´
edgement to the Spanish Fundacion Ramon Areces for a post-
doctoral fellowship.
Analytical Whole-Cell Biotransformations
Inhibition Studies: Biotransformations were performed at a
25 mL analytical scale in 250 mL Erlenmeyer flasks, using a re-
ciprocal agitated water bath maintained at 308C. They were
followed by GC analysis of samplings.
Inhibition levels of ketone 1 were determined by addition of
1 (dissolved in the minimum amount of ethanol) at a final con-
centration of 0.3, 0.5, 1.0 and 1.3 g Lꢀ1, respectively, to a E. coli
TOP10[pQR239] culture at pH 7. Nearly total conversion of 1
was observed at the initial ketone concentrations lower than
1.3 g Lꢀ1. Some 25% of the compound 1 remained after 24 h
of biotransformation at higher concentration.
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1058
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