Enzymatic Baeyer-Villiger oxidation of bicyclic diketones

Article abstract of DOI:10.1002/adsc.200505027

Cyclohexanone monooxygenase from Acinetobacter calcoaceticus was employed for the Baeyer-Villiger oxidation of racemic bicyclic diketones such as the Wieland-Miescher and the Hajos-Parrish diketones and of some of their derivatives. The corresponding lactones were produced in a highly regio-and enantioselective manner. The reactions were carried out using a crude enzyme preparation in aqueous buffer, at room temperature, and the recycling of the expensive coenzyme NADPH was conducted with a second ancillary enzymatic system. The enzymatic process was simple and easy to handle, thus providing a very practical tool to access enantiopure lactones.

Full text of DOI:10.1002/adsc.200505027

FULL PAPERS
Enzymatic Baeyer–Villiger Oxidation of Bicyclic Diketones
Gianluca Ottolina,a,* Gonzalo de Gonzalo,a Giacomo Carrea,a Bruno Danielib
a
Istituto di Chimica del Riconoscimento Molecolare, CNR ,Via Mario Bianco 9, 20131 Milano, Italy
Fax: (þ39)-02-2850-0036, e-mail: gianluca.ottolina@icrm.cnr.it
b
`
Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
Received: January 20, 2005; Accepted: April 1, 2005
Abstract: Cyclohexanone monooxygenase from Aci- buffer, at room temperature, and the recycling of the
netobacter calcoaceticus was employed for the Baey- expensive coenzyme NADPH was conducted with a
er–Villiger oxidation of racemic bicyclic diketones second ancillary enzymatic system. The enzymatic
such as the Wieland–Miescher and the Hajos–Parrish process was simple and easy to handle, thus providing
diketones and of some of their derivatives. The corre- a very practical tool to access enantiopure lactones.
sponding lactones were produced in a highly regio-
and enantioselective manner. The reactions were car- Keywords: Baeyer–Villiger oxidation; diketones; en-
ried out using a crude enzyme preparation in aqueous zyme catalysis; lactones; monooxygenase
Introduction
The chemical Baeyer–Villiger oxidation of bicyclic
and polycyclic ketones has been employed quite often
The enzymatic Baeyer–Villiger oxidation is an interest- to generate the corresponding lactones, since these
ing methodology aiming at obtaining optically active structures are frequently found in natural products or
lactones and esters in environmental friendly processes, are key intermediates in synthesis.[6] However, the pres-
avoiding the conventional use of peracids or peroxides ence of two (or more) non-equivalent keto groups in the
and heavy-metal catalysts. Among the various Baeyer– molecule raised problems of selectivity with the forma-
Villiger monooxygenases, the most studied to date is tion of mixtures of products.[7]
the flavoprotein cyclohexanone monooxygenase
This difficulty could, in principle, be surmounted by
(CHMO) from Acinetobacter calcoaceticus NCIMB exploring the recognized chemo-, regio- and enantiose-
9871,[1] and many examples of its substrate selectivity lectivity of enzymes and in this paper we have investigat-
and enantioselectively to oxidize ketones, sulfides, di- ed the ability of CHMO to carry out the Baeyer–Villiger
thioacetals and amines,[2] sometimes on a preparative oxidation of two representative bicyclic diketones, the
scale,[3] have been reported. The reaction takes place Wieland–Miescher[8] (1) and the Hajos–Parrish[9] (12)
in aqueous buffer using atmospheric O2 as the oxidant diketones and of some of their derivatives, which are
and whole cells or isolated enzyme as the catalyst. In building blocks for the synthesis of many natural prod-
the latter case, the oxidation has to be coupled with a ucts.[10]
second enzymatic reaction for the regeneration of the
expensive cofactor NADPH.
The use of enzymes in synthesis has two major hurdles Results and Discussion
to overcome, their availability and their scarce substrate
versatility. Until some years ago the number of Baeyer– The enzymatic Baeyer–Villiger oxidations were carried
Villiger monooxygenases was limited and only recently out using as the biocatalyst a crude preparation of re-
is their accessibility from different sources increasing.[2f] combinant CHMO from Escherichia coli TOP10
On the other hand, despite the fact that many molecules pQR239[11] and were coupled to an ancillary enzymatic
with different skeletons have been tested, still only few reaction in order to regenerate NADPH. As the
studies on substrates with more than one reactive group NADPH regeneration system, glucose 6-phosphate
have been conducted. For instance, CHMO converted 3- and glucose 6-phosphate dehydrogenase (G6PDH,
and 4-thiacyclohexanones exclusively into the lactone Scheme 1) were employed.[12]
products with no detectable sulfide oxidation,[4] and
Oxidation of the racemic Wieland–Miescher ketone
the same happened with 4-methylamine-cyclohexa- (rac-1) with the crude enzyme preparation gave the lac-
none, where the lactone was formed with no N-oxida- tone (S)-2 in 35% yield and in enantiomerically pure
tion.[5]
form, as demonstrated by chiral HPLC analysis. Oxygen
insertion at the C1–C8a carbon-carbon bond of (S)-1
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Gianluca Ottolina et al.
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Scheme 1.
was inferred by NMR analysis, which revealed the ab-
Furthermore, by pushing the conversion of rac-1 close
sence of both the -OCH2- group (which rules out oxygen to 50% in lactone, the remaining ketone (R)-1 was ob-
insertion at the C1–C2 or C6–C7 carbon-carbon bond) tained with an ee of 96% (yield 40%). Therefore, due
and the -OCHC- fragment (which rules out oxygen in- to the characteristics of this enzymatic preparation, it
sertion at the C5–C6 carbon-carbon bond). This oxygen is possible to discriminate between the (S) enantiomer
insertion is in agreement with previous studies by Walsh of 1 that is oxidized to the lactone, and the (R) enantiom-
and Chen which have shown that CHMO is not able to er, which remains almost unaffected. In principle, this
oxidize a,b-unsaturated ketones.[4] The absolute config- might be an alternative methodology for the prepara-
uration of the lactone product was deduced by the enzy- tion of (R)-1 which, in analogy with the chemical syn-
matic oxidation of the enantiopure ketone (S)-1 which thesis of (S)-1, could be accessible via proline-enamine
gave the lactone (S)-2. The unreacted starting material, Robinson annulation reaction of 1-methylcyclohex-
recovered in 43% yield (Scheme 1), was shown to be the ane-1,3-dione with methyl vinyl ketone, employing the
ketone (R)-1, ee 80%, by HPLC comparison with an au- expansive d-proline [(R)-(þ)-proline] as catalyst and te-
thentic sample. In addition, careful inspection of the chi- dious repeated crystallization at low temperature.[14]
ral HPLC profile of this reaction revealed the presence
Starting from the Wieland–Miescher ketone (rac-1),
of small amounts of by-products, about 5% altogether, racemic 8a–methylhexahydronaphthalene-1,6-dione 5
that were identified as the alcohols derived by reduction was easily obtained by selective hydrogen transfer em-
of the saturated carbonyl. Since under this enzymatic ploying limonene and 10% Pd/C. The stereoselectivity
conditions, the only product obtained from (S)-1 was for this type of hydrogenation has been demonstrated
the lactone (S)-2, these alcohols could only derive by a to be almost exclusively in favor of the formation of
slow enzymatic side reaction on the (R)-1 ketone. Ac- the cis-isomer.[15] The treatment of rac-cis-5 with crude
tually, the incubation of (R)-1 with a crude preparation CHMO gave the enantiopure lactone (5aR,9aS)-6 (yield
of CHMO gave a small amount of alcohols (4aR,5S)-3 22%, ee ꢀ99%, Scheme 2).
and (4aR,5R)-4 identified by comparison with authentic
samples obtained via NaBH4 reduction.[13]
Oxygen insertion at the C1–C8a carbon-carbon bond
ꢁ
ꢁ
was deduced by the absence of OCH2 signals in the
NMR spectra. It should be emphasized that only one
out of four possible regioisomers was formed by enzy-
matic oxidation. Besides the lactone, the second reac-
tion product was an alcohol isolated with 32% yield
and ee ꢀ99%, identified as (4aS,6S,8aR)-7 and derived
The formation of the two alcohols 3 and 4 was due to a
contaminating dehydrogenase activity, present in the
crude preparation of CHMO, which came from the E.
coli strain where the enzyme was overexpressed.[11] In
fact, when rac-1 was treated with a crude preparation
obtained from the same E. coli strain, but that did not
contain the CHMO gene, only the alcohols were formed
in small amounts. It should be mentioned that when a
purified preparation of CHMO devoid of contaminating
dehydrogenase was employed, only the lactone (S)-2
was formed.
Scheme 2.
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Enzymatic Baeyer–Villiger Oxidation of Bicyclic Diketones
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Scheme 3.
by the complete reduction of (4aS,8aR)-5 by contami-
Neither ketone (S)-12 nor (1aS,4aS,7aS)-13 and
nant dehydrogenase(s). It should be mentioned that (3aS,7aS)-11 were affected by CHMO (Scheme 4),
the rate of reduction of (4aS,8aR)-5 by dehydrogen- that was quite surprising since the structurally closely re-
ase(s) was even faster than that of oxidation of lated compounds 1 and 8 with the same absolute config-
(4aR,8aS)-5 by CHMO. The absolute stereochemistry uration were oxidized by CHMO to lactones.
of products 6 and 7 was inferred by conducting the enzy-
Taken all together, these results suggest that among
matic reaction with the single enantiomers, which were the compounds investigated in this work the structure
obtained by chemical reduction of the respective opti- able to better fit the active site of CHMO is that of dike-
cally active ketones 1.
toperhydronaphthalene in the (S) configuration. The
Hydrogen peroxide oxidation of rac-1 gave the keto- fact that the diketoperhydroindene derivatives with
epoxide rac-8, which is formed through an anti attack the same absolute configuration do not react is hardly
to the conjugated double bond with respect to the meth- compatible with the hypothesis that substrates cannot
yl group (Scheme 3).[16]
be accommodated in the active site.[17] A possible ex-
The enzymatic treatment of rac-8 gave the enantio- planation might be that the five-membered ring does
pure lactone (1R,3S,7S)-9, again through the selective not possess the conformational mobility endowed with
Baeyer–Villiger oxidation of the carbonyl in position the six-membered ring that allows the latter to satisfy
5. The unreacted starting material was recovered, as the stereoelectronic requirements essential for the sub-
the (1aR,4aR,8aR)-8 enantiomer, in high optical purity sequent migration in the Criegee intermediate. In fact,
(ee 95%). (1aR,4aR,8aR)-8 and (1aS,4aS,8aS)-8, pre-
pared from the corresponding enantiomers of 1, con-
firmed that only the latter was oxidized by CHMO, leav-
ing the other enantiomer unreacted. As depicted in
Scheme 3, another reaction product was isolated and
identified as the alcohol 10 by usual spectroscopic anal-
ysis. In fact the 1H NMR spectrum reveals H-4 as a sin-
glet at 3.05 ppm due to an almost orthogonal relation-
ship to H-3. This oxymethine proton is in turn a dd for
the vicinal coupling to the C-2 protons which resonate
in the 1.27–1.40 and 1.80–2.10 ppm zones, respectively,
and are connected to the same carbon at 28.52 ppm.
Time course experiments showed the slow formation
of 10 by reduction of 9 due to contaminant dehydrogen-
ase(s).
The Hajos–Parrish ketone 12 permitted us to gain
some information on the difference in enzyme reactivity
between the diketoperhydronaphthalene and the dike-
toperhydroindene structures. Starting from the com-
mercially available enantiopure ketol (3aS,7aS)-11,
compound (S)-12 and its epoxide derivative
(1aS,4aS,7aS)-13 were easily prepared and then treated
with CHMO (Scheme 4).
Scheme 4.
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Gianluca Ottolina et al.
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Scheme 5.
the saturated ketone (3aR,7aS)-14, obtained by reduc- were accepted as substrates (e.g., 1 and 5 versus 12 and
tion of (S)-12 with limonene and 10% Pd/C,[18] was 14).
oxidized by CHMO to lactone (5aS,8aS)-15 in low
The reactions were carried out using a crude enzyme
yield (Scheme 5), as demonstrated by NMR anal- preparation obtained from E. coli cells in which the
ysis, that showed the presence of the signals of two gene of CHMO had been overexpressed. It was chosen
doublets of doublets for the oxymethylene of the to utilize a crude biocatalyst because it was more con-
ꢁ
ꢁ
ꢁ
OCH2 CH fragment.
venient and required less man-power to obtain it. These
Other subtle conformational and sterical factors are important requisites in view of possible future large-
might play an important role in stirring the reaction out- scale applications of CHMO. However, it should be
come. Indeed, it is interesting to note that ketone (S)-17, mentioned that the crude enzyme contained contami-
obtained from (S)-12 via NaBH4 reduction to 16[19] and nating dehydrogenase activities which, as in the case of
subsequent oxidation,[20] now proved to be a substrate 5, completely reduced one of the enantiomers to a
for CHMO, forming lactones (S)-18 (yield 82%) by in- keto alcohol. Since the keto alcohol was optically pure,
sertion of the oxygen at the C1–C7a carbon-carbon the result might even be considered positive, should
bond (Scheme 5).
one need this product. Anyway, as shown in the case of
compound 1, this drawback can be easily overcome by
using CHMO purified by conventional methods.
Conclusion
CHMO proved to be able to catalyze the Baeyer–Villig-
er oxidation of some racemic diketones in a highly regio-
and enantioselective manner, yielding lactones with
very high optical purity. In fact the ee of the products
was ꢀ99% which points to an enantiomeric ratio for
CHMO higher than one hundred. The lactones obtained
might be valuable synthons for fine and medicinal chem-
istry because of their degree of complexity and proper
functionalization. As already seen with other substrates
(monoketones, sulfides, organic sulfites),[2] enzyme se-
lectivity is quite dependent on substrate structure (e.g.,
see the different regiopreference shown with substrate
5 compared to substrate 14). A marked dependence on
substrate structure was also observed for the catalytic ef-
ficiency of the enzyme, which sometimes was totally
Experimental Section
General Remarks
Melting points (uncorrected) were determined with a Reich-
ert-Kçfler apparatus. IR spectra were recorded on a Jasco
FTIR 610. Optical rotations were determined on a Perkin El-
mer 141 polarimeter. Flash chromatography: silica gel 60
(70–230 mesh, Merck). Chiral HPLC analysis: Jasco HPLC in-
strument (model 880-PU pump, model 870-UV/VS detector)
equipped with a Chiracel OD (Daicel) chiral column. 1H and
13C NMR spectra at 300 MHz and 72.5 MHz were recorded
in CDCl3 on a Bruker AC-300. Mass spectra were recorder
on a GC-MS-EI (Finnigan-Thermo). All chiral compounds
inactive with compounds closely similar to those that were purchased from Aldrich-Fluka.
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Enzymatic Baeyer–Villiger Oxidation of Bicyclic Diketones
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1.55 (2 H, m), 1.68–1.75 (1 H, dq, J¼19.03, 2.65 Hz), 1.78–
1.86 (1 H, dquint, J¼12.45, 3.65 Hz), 1.88–2.00 (3 H, m),
2.19–2.34 (3 H, m), 3.6 (1 H, m); 13C NMR d¼211.00, 70.88,
48.40, 43.53, 38.36, 37.82, 32.94, 32.72, 26.57, 26.27, 22.06.
Enzymatic Oxidation of Wieland–Miescher Ketone (1)
The Wieland–Miescher ketone (1; 100 mg, 0.56 mmol) was
magnetically stirred at room temperature in potassium phos-
phate buffer (50 mM, pH 8.7, 22 mL), containing NADP
(2.78 mg, 3.6 mmol),
glucose
6-phosphate
(188 mg,
0.62 mmol), CHMO (10 U) and G6PDH (50 U). The reaction
was stopped after 26 hours and worked up. The crude product
was purified by flash chromatography (eluent ethyl acetate-
light petroleum ether, 2:1) to give (R)-(ꢁ)-1; yield: 43 mg
(43%); ee 80%; [a]2D5: ꢁ67.0 (c 2, benzene) and (S)-(þ)-2; yield:
39 mg (35%); ee ꢀ99%; white powder; [a]2D5: þ90.3 (c 1.5,
MS: m/z¼194 (Mþ, 9), 176 (21), 122 (36), 108 (71), 93 (90);
1H NMR: d¼1.64 (3 H, s), 1.83–1.93 (1 H, m), 2.12–2.22
(1 H, m), 2.25–2.32 (1 H, m), 2.43–2.74 (7 H, m) 5.9 (1 H, s);
13C NMR d¼196.35, 171.95, 127.00, 111.19, 81.23, 35.40,
35.28, 32.50, 28.97, 26.08, 23.52; HPLC: column Chiracel OD
Diacel, l245, flow rate 1 mL/min, eluent: 93% light petroleum
ether, 7% i-PrOH; Rt: (4aR,5R)-4 12.5 min, (4aR,5S)-3
14.0 min, (S)-(þ)-1 16.8 min, (R)-(ꢁ)-1 18.4 min, (S)-(þ)-2
42.0 min.
Enzymatic Oxidation of rac-8
Compound rac-8[16] (120 mg, 0.61 mmol) was magnetically stir-
red at room temperature in potassium phosphate buffer
(50 mM, pH 8.7, 25 mL), containing NADP (3 mg, 3.9 mmol),
glucose 6-phosphate (207 mg, 0.67 mmol), CHMO (12 U)
and G6PDH (60 U). The reaction was stopped after 24 hours
and worked up. The crude product was purified by flash chro-
matography (eluent ethyl acetate-light petroleum ether, 3:1)
to give 42.5 mg of starting material (1aR,4aR,8aR)-8, 33.5 mg
of 9 and 14 mg of 10.
chloroform); mp 140–1438C; IR (nujol): n¼1720, 1659 cmꢁ1
;
(1aR,4aR,8aR)-8: Yield: 35%; [a]2D5: þ188.2 (c 2, chloro-
form); ee 95%; HPLC: column Chiracel OD Diacel, l215, flow
rate 1 mL/min, eluent: 90% light petroleum ether, 10% i-
PrOH; Rt: (1aS,4aS,8aS)-8 10.0 min and (1aR,4aR,8aR)-(þ)-8
11.4 min.
(1R,3S,7S)-7-Methyl-2,8-dioxatricyclo[5.5.0.01,3]dodecane-
Compounds (4aR,5S)-3 and (4aR,5R)-4 were confirmed by
comparison with authentic samples.[13]
4,9-dione (9): Yield: 26%; oil; IR (nujol): n¼1713, 1289 cmꢁ1
;
ee ꢀ99%; MS: m/z¼182 (2), 154 (4), 139 (15), 111 (42), 99
(100); 1H NMR: d¼1.54 (1 H, dt, J¼14.9, 4.1 Hz), 1.65 (3 H,
s), 1.84 (1 H, dd, J¼11.7, 7.8 Hz), 1.95–2.05 (2 H, m), 2.28
(1 H, ddd, J¼13.6, 11.7, 5.8 Hz), 2.5–2.65 (3 H, m), 2.73
(1 H, ddd, J¼13.5, 9.1, 3.8 Hz), 3.02 (1 H, ddd, J¼13.5, 8.1,
3.6 Hz), 3.23 (1 H, s); 13C NMR: d¼206.50, 172.06, 83.05,
71.40, 63.94, 35.96, 33.69, 30.62, 29.14, 23.16, 19.46; HPLC: col-
umn Chiracel OD Diacel, l215, flow rate 1 mL/min, eluent: 95%
light petroleum ether, 5% i-PrOH; Rt: (1R,3S,7S)-9 45.4 min.
(1S,3R,4S,7R)-4-Hydroxy-7-methyl-2-oxatricy-
Chemical Oxidation of Wieland–Miescher Ketone (1)
The Wieland–Miescher ketone (1; 70 mg, 0.39 mmol) was mag-
netically stirred at room temperature in dichloromethane
(7.8 mL) containing mCPBA (406 mg, 1.18 mmol). The reac-
tion was stopped after 48 hours and worked up. The crude
product was purified by flash chromatography (eluent ethyl
acetate-light petroleum ether, 2:1) to give 2; yield: 72 mg
(95%). HPLC: column Chiracel OD Diacel, l245, flow rate
1 mL/min, eluent: 93% light petroleum ether, 7% i-PrOH;
Rt: (S)-(þ)-2 42.0 min, (R)-(ꢁ)-2 54.2 min.
clo[5.5.0.01,3]dodecan-9-one (10): Yield: 11%; white powder;
mp 83–858C; [a]2D5: ꢁ18.3 (c 1, chloroform); ee ꢀ99%; IR (nu-
jol): n¼3420, 1704, 1295 cmꢁ1; MS: m/z¼212 (Mþ, 4), 194 (3),
1
165 (4), 111 (38), 97 (100); H NMR: d¼1.27–1.40 (2 H, m),
1.63 (1 H, ddd, J¼9.95, 1.8, 1.53 Hz), 1.7 (3 H, s), 1.8–2.1
(4 H, m), 2.43 (1 H, ddd, J¼14.9, 11.85, 4.1 Hz), 2.74 (1 H,
ddd, J¼15.82, 12.62, 2.95 Hz), 2.95 (1 H, dd, J¼13.0, 6.6 Hz),
3.05 (1 H, s), 4.08 (1 H, dd, J¼9.7, 7.06 Hz).
Enzymatic Oxidation of rac-cis-5
Compound rac-cis-5[15] (110 mg, 0.61 mmol) was magnetically
stirred at room temperature in potassium phosphate buffer
(50 mM, pH 8.7, 24.4 mL), containing NADP (3 mg,
3.9 mmol), glucose 6-phosphate (204 mg, 0.67 mmol), CHMO
(11 U) and G6PDH (55 U). The reaction was stopped after
26 hours and worked up. The crude product was purified by
flash chromatography (eluent ethyl acetate-light petroleum
ether, 1:1) to give 29 mg of starting material 5 (ee 4%), [a]2D5:
ꢁ0.3 (c 1, chloroform), 27 mg of 6 and 36 mg of 7.
Chemical Oxidation of rac-8
Compound rac-8[16] (25 mg; 0.13 mmol) was magnetically stir-
red at room temperature in dichloromethane (2.5 mL) and
mCPBA was added portionwise over 3 hours (100 mg;
0.38 mmol). After 24 hours the reaction mixture was quenched
by the addition of sodium dithionite and worked up. The crude
product was purified by flash chromatography (eluent ethyl
acetate-light petroleum ether, 3:1) to give racemic 9; yield:
8 mg (30%). HPLC: column Chiracel OD Diacel, l215, flow
rate 1 mL/min, eluent: 95% light petroleum ether, 5% i-
PrOH; Rt: (1S,3R,7R)-9 43.4 min, (1R,3S,7S)-9 45.4 min.
(5aR,9aS)-9a-Methylhexahydrobenzo[b]oxepine-2,7-dione
(6): Yield: 22%; white powder; [a]2D5: þ16.0 (c 1.5, chloroform);
ee ꢀ99%; mp 70–738C; IR (nujol): n¼1716 cmꢁ1; MS: m/z¼
196 (Mþ, 3), 178 (5), 168 (5), 125 (37), 99 (100); 1H NMR: d¼
1.22 (3 H, s), 1.65–1.70 (3 H, m), 1.75–1.85 (4 H, m), 2.67–
2.74 (3 H, m), 2.85–2.95 (3 H, m); 13C NMR: d¼210.02,
174.18, 80.15, 43.13, 41.22, 41.00, 37.20, 37.07, 29.62, 24.40,
17.43.
(4aS,6S,8aR)-6-Hydroxy-8a–methyloctahydronaphthalen-
1-one (7): Yield: 32%; oil; [a]2D5: þ47.5 (c 2, chloroform); ee ꢀ
99%; IR (nujol): n¼3474, 1687 cmꢁ1; MS: m/z¼182 (Mþ, 5),
Enzymatic Oxidation of (3aR,7aS)-14
Compound (3aR,7aS)-14[18] (76 mg; 0.45 mmol) was magneti-
cally stirred at room temperature in potassium phosphate buf-
fer (50 mM, pH 8.7, 12.5 mL), containing NADP (2.3 mg;
1
164 (58), 121 (100), 93 (88),79 (56); H NMR: d¼0.96 (1 H,
dt, J¼13.7, 4.0 Hz), 1.22 (3 H, s), 1.24–1.43 (3 H, m), 1.47–
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Gianluca Ottolina et al.
FULL PAPERS
3 mmol), glucose 6-phosphate (153 mg, 0.5 mmol), CHMO
(7.6 U) and G6PDH (38 U). The reaction was stopped after
24 hours and worked up. The crude product was purified by
flash chromatography (eluent ethyl acetate-light petroleum
ether, 3:1) to give 15; yield: 11.3 mg (14%); white powder;
[a]2D5: þ59.3 (c 0.7, chloroform); mp 76–798C; IR (nujol):
n¼1733 cmꢁ1; MS: m/z¼182 (Mþ, 20), 154 (15), 123 (48), 68
Carrea, R. Wohlgemuth, R. Furstoss, J. M. Woodley,
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1
(100); H NMR d¼1.17 (3 H, s), 1.55 (1 H, ddd, J¼11.45,
9.5, 2.0 Hz), 1.85–1.95 (1 H, m), 1.98 (1 H, ddd, J¼11.45,
10.3, 2.0 Hz), 2.1–2.25 (1 H, m; 1 H, m, X part of an ABX sys-
tem), 2.3–2.5 (2 H, m), 2.56 (1 H, ddd, J¼14.8, 9.4, 2.0 Hz),
2.66 (1 H, ddd, J¼14.8, 10.3, 2.0 Hz), 4.18 (1 H, dd, J¼13.5,
4.2 Hz, B part of an ABX system), 4.37 (1 H, dd, J¼13.5,
1.8 Hz, A part of an ABX system); 13C NMR: d¼210.50,
172.51, 67.10, 45.95, 44.50, 34.89, 29.39, 26.74, 21.64, 20.63.
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Enzymatic Oxidation of (S)-17
Compound 17[20] (47 mg, 0.31 mmol) was magnetically stirred
at room temperature in potassium phosphate buffer (25 mM,
pH 8.7, 12.5 mL), containing NADP (1.6 mg, 2 mmol), glucose
6-phosphate (105 mg, 0.34 mmol), CHMO (4.7 U) and
G6PDH (24 U). The reaction was stopped after 8 hours and
worked up. The crude product was purified by flash chroma-
tography (eluent ethyl acetate-light petroleum ether, 3:1) to
give 18; yield: 42.5 mg (82%); oil; MS: m/z¼166 (Mþ, 39),
151 (40), 123 (29), 106 (53); 91 (100); 1H NMR: d¼1.64 (3 H,
s), 1.5–1.8 (4 H, m), 1.8–2.2 (2 H, m), 2.25–2.75 (4 H, m),
4.83 (1 H, bs); 13C NMR: d¼172.32, 127.21, 111.30, 80.53,
31.26, 29.70, 28.83, 26.31, 23.00, 19.71.
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Acknowledgements
We thank the Biotechnology Programme of the European Com-
mission (QLK3-CT-2001-00403) for financial support. G.d.G.
thanks FICYT (Principado de Asturias, Spain) for a postdoctor-
al fellowship.
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