Chemoenzymatic and yeast-catalysed synthesis of diastereomeric ethyl γ-phenyl and γ-(n-pyridyl)paraconates

Article abstract of DOI:10.1016/j.tetasy.2008.08.011

The synthesis of γ-phenyl and γ-(n-pyridyl)paraconates was accomplished by chemical reduction of their respective ketodiester precursors followed by cyclisation of the resulting hydroxy diester intermediates. The cis- and trans-lactones thus obtained were separated and separately subjected to enzymatic hydrolysis with HLAP. The cis-lactonic esters had enantiomeric excesses ranging from 94% to 99%, while for the trans-isomers the ee's ranged from 80% to 93%. The same ketodiester precursors were subjected to reduction with a series of yeasts. The absolute configuration of trans-(-)-2-pyridyl paraconic acid was assigned by means of X-ray analysis of its hydrobromide salt, while the absolute configurations of the other lactones were determined via analysis of their respective CD curves.

Full text of DOI:10.1016/j.tetasy.2008.08.011

Tetrahedron: Asymmetry 19 (2008) 2026–2036
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemoenzymatic and yeast-catalysed synthesis of diastereomeric
ethyl
c-phenyl and c-(n-pyridyl)paraconates
a
a
a
a,
*
Cristina Forzato a, , Giada Furlan , Patrizia Nitti , Giuliana Pitacco , Ennio Valentin
,
*
Ennio Zangrando a, Pietro Buzzini b, Marta Goretti b, Benedetta Turchetti b
a Dipartimento di Scienze Chimiche, Università di Trieste, via Licio Giorgieri 1, I-34127 Trieste, Italy
b Dipartimento di Biologia Applicata, Sezione di Microbiologia, Università di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of c-phenyl and c-(n-pyridyl)paraconates was accomplished by chemical reduction of their
Received 24 June 2008
Accepted 7 August 2008
Available online 6 September 2008
respective ketodiester precursors followed by cyclisation of the resulting hydroxy diester intermediates.
The cis- and trans-lactones thus obtained were separated and separately subjected to enzymatic hydro-
lysis with HLAP. The cis-lactonic esters had enantiomeric excesses ranging from 94% to 99%, while for the
trans-isomers the ee’s ranged from 80% to 93%. The same ketodiester precursors were subjected to reduc-
tion with a series of yeasts. The absolute configuration of trans-(ꢀ)-2-pyridyl paraconic acid was assigned
by means of X-ray analysis of its hydrobromide salt, while the absolute configurations of the other
lactones were determined via analysis of their respective CD curves.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
applications, especially in the perfume industry and solvent
production.4
5-Oxo-tetrahydro-3-furancarboxylic acid derivatives, com-
monly known as paraconic acid derivatives, constitute a small class
Besides the presence of a carboxylic group at the b-carbon atom,
naturally occurring paraconic acid derivatives also bear an alkyl
of highly substituted
c
-butyrolactones, which are metabolites of
chain at the
group at the a-position. Examples of natural paraconic acid deriv-
c
-carbon atom and a methyl1e,5 or a methylene1f,g
mosses, lichens and fungi.1 The first member of the series, paracon-
ic acid 1, namely 5-oxo-tetrahydro-3-furancarboxylic acid (Fig. 1),
is non-natural, but it is an important intermediate in the synthesis
of A-factor,2 an inducer of the biosynthesis of streptomycin in
inactive mutants of Streptomyces griseus, first isolated by Khokhlov
et al.3 in 1973. The esters of paraconic acid have industrial
atives are rocellaric acid (ꢀ)-2,6 phaseolinic acid (ꢀ)-37 and methy-
lenolactocin (ꢀ)-4a8 (Fig. 1), while terebic acid (ꢀ)-5, 2-methyl
paraconic acid (ꢀ)-6 and 4-methyl paraconic acid (ꢀ)-7 and their
corresponding esters are examples of non-natural compounds,
whose syntheses have been accomplished in our laboratories.9
HOOC
HOOC
C13H27
HOOC
HOOC
C5H11
O
R
O
O
O
O
O
O
O
paraconic acid
phaseolinic acid
rocellaric acid
(—)-2
4a: R = C5H11: methylenolactocin
4b: R = C8H17: C75
1
(—)-3
HOOC
HOOC
HOOC
O
O
O
O
O
O
terebic acid
(—)-5
(—)-6
(—)-7
Figure 1. Examples of natural and non-natural paraconic acid derivatives.
* Corresponding authors. Tel.: +39 040 5583921; fax: +39 040 5583903 (C.F.).
E-mail address: cforzato@units.it (C. Forzato).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.08.011

C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
2027
Recently, the unnatural compound 4b, labelled C75,10 was
found to be an inhibitor of mammalian type I fatty acid synthase
at the b-ketoacyl synthase (KAS) domain. It has been shown to
have significant in vivo antitumour activity against human breast
cancer xenografts.11
when hydrolases can be used, owing to their easy accessibility and
low cost. This is the case for lactonic esters which have been suc-
cessfully desymmetrised by this method.5b,9a,b
2. Results and discussion
Within the frame of our studies on the synthesis of paraconic
acid derivatives in diastereo- and enantiomerically pure forms,
2.1. Synthesis of racemic substrates
we turned our attention to
c-phenyl and c-heteroaryl paraconic
acids, which are uncommon in the literature. The heteroaryl group,
2-pyridyl, 3-pyridyl and 4-pyridyl were chosen: in fact these par-
The synthetic procedure leading to racemic cis- and trans-
c-
aryl-b-ethoxycarbonyl- -butyrolactones 8a–d and 9a–d, respec-
c
ticular
8a–d and 9a–d, respectively (Fig. 2), seemed interesting as poten-
tially biologically active compounds. For example, -pyridyl
-butyrolactones have been shown to exhibit antihelminthic
properties.12
c-butyrolactones, present as cis- and trans-diastereomers
tively, involves the corresponding ketodiesters 10a–d as interme-
diates (Scheme 1). As shown in Scheme 1, 10a18 was prepared by
the reaction of ethyl benzoylacetate with ethyl bromoacetate un-
der basic conditions,19 while 10b, 10c and 10d were synthesised
by a Claisen condensation between diethyl succinate and the cor-
responding ethyl 2-, 3-, and 4-pyridylcarboxylates under basic con-
ditions.12 Subsequent reduction of the carbonyl group of 10a–d
with sodium borohydride afforded the corresponding hydroxy
diesters 11a–d and 12b,d, as syn- and anti-diastereomers, respec-
tively, in an admixture with their cyclisation products 8a–d and
9a–d. The reduction products 11a–d and 12b,d were not isolated
but only identified in their respective crude reaction mixtures.
Their cyclisation into the target molecules was accomplished
either by refluxing in toluene, in the presence of PTSA as the cata-
lyst, or by microwave irradiation. This latter procedure was neces-
sary in particular for the 4-pyridyl derivative 11d which was hard
to cyclise.
c
c
EtOOC
Ar
Ar
Ph
2-Py
3-Py
4-Py
a
b
c
d
O
O
cis: 8a-d
trans: 9a-d
Figure 2. Ethyl c-phenyl and c-heteroarylparaconates 8a–d and 9a–d.
Within the biochemical methodologies which find frequent
applications, also for the production of fine chemicals, are those
which make use of microbial cells (e.g., yeast cells) as biocata-
lysts.13 As an example, connected to the molecules presented in
this work, the bioreduction of simple heteroaromatic ketones, such
as 2-, 3- and 4-acetylpyridine by Rhodococcus sp.,14 to the corre-
sponding alcohols can be cited. These compounds are important
building blocks for the synthesis of a wide range of pharmaceuti-
cals, including the alkaloids akuamidine and heteroyohimidine,15
and other biologically active compounds.
However, when the substrate contains different functionalities,
the use of microbial whole cell systems can sometimes lead to
undesired by-products, owing to the presence of various enzymes
(located at the cytoplasm level or, more frequently, associated with
some wall or membrane components) capable of catalysing side
reactions, such as hydrolysis of an ester group,16 accompanied by
decarboxylation.17 Moreover, one of the major problems in using
whole cell yeasts is the presence of numerous alcohol dehydrogen-
ases, which can be either (R)- or (S)-selective and hence make their
use in organic synthesis difficult (especially when repeating exper-
iments with a new batch of a yeast). For these reasons, the use of
purified enzymes is often regarded as more profitable, in particular
The
c-phenyl and c-heteroaryl-c-butyrolactones prepared in
this manner were mixtures of diastereomers, in which the cis-
isomers 8a–d prevailed over the trans-ones 9a–d, their ratios,
however, were dependent upon the time required for the lactoni-
sation reaction to go to completion. After basic equilibration with
DBU, all mixtures consisted of 80% of the trans-isomers and 20%
of the cis-isomers. The mixtures were separated on column chro-
matography, and each isomer was subjected to enzymatic
hydrolysis.
The geometry of the c
-phenyl derivatives 8a and 9a is known.20
The assignment of the correct geometry to the other diastereo-
meric pairs followed from the results of the equilibration with
DBU and from a comparison of the chemical shifts of H-2 and
H-3 in their respective 1H NMR spectra (Table 1). In all cases, the
signals of the trans-substituted lactones resonated at higher field
than those of the cis-isomers, in accordance with the literature
data.20,21
Within the synthetic route leading to the desired optically
active
c-lactones, the enantiodifferentiating process was applied
either on the ketodiester precursors 10a–d, by yeast reduction, or
COOEt
COOEt
Br
COOEt
Ph
n-Py
OEt
NaH
Et2O
COOEt
Ar
COOEt
+
EtONa/EtOH
O
O
r.t.
Δ, 1h
COOEt
O
10a-d
NaBH4 (0.6 eq)
EtOH, 0 ºC, 1h
COOEt
COOEt
EtOOC
Ar
EtOOC
PTSA
Ar
Ph
2-Py
3-Py
4-Py
Ar
+
toluene
a
b
c
d
O
Δ
Ar
O
O
8a-d
O
OH
9a-d
syn-11a-d
anti-12[a],b,[c],d
Scheme 1. Synthesis of racemic 8a–d and 9a–d.

2028
C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
Table 1
were only identified spectroscopically. The most successful reac-
tion was subsequently repeated at large-scale level in order to be
able to purify the product.
Significant 1H NMR data for the diastereomeric pairs 8a–d and 9a–d
c
-Lactone
H-2
d, ppm
H-3
d, ppm
Reductions of the ketodiesters 10a–d catalysed by the nine
yeast strains were complicated by the high hydrolytic activity
exhibited by all strains and by the decarboxylation reactions occur-
ring on many intermediates. Scheme 2 summarises the possible
pathways of the transformations involved, based on the nature of
the products identified. It should be noted that the products con-
taining a carboxy group have been identified as their respective
methyl esters, although for the sake of clarity, they appear in
Scheme 2 as carboxylic acid derivatives. In Section 4, they are given
as methyl esters.
8a
9a
8b
9b
8c
9c
8d
9d
5.76
5.66
5.76
5.75
5.79
5.67
5.71
5.66
3.72
3.32
3.82
3.80
3.77
3.34
3.77
3.26
Pathway a, which is the most common for all yeast strains, in-
volves the hydrolysis of the secondary ester group of 10a–d to give
the corresponding hemiesters 13a–d, whose subsequent fate is
on the final products 8a–d and 9a–d, by enzymatic hydrolysis of
their ester group.
decarboxylation and hydrolysis with formation of the c-ketoacids
2.2. Yeast-catalysed bioreductions of ketodiesters 10a–d
14a,[b],c,d. In two cases, namely for 14b, not identified, and 14d,
further reduction of the carbonyl group afforded the corresponding
hydroxy acids 15b and 15d, the former also found as its lactonisa-
tion product, 16b whose ee could not be evaluated.
Pathway b, which involves the hydrolysis of the primary ester
group and reduction of the carbonyl group of the ketodiester, is
followed only by compounds 10c and 10d, to give hydroxy
The ketodiesters 10a–d were submitted to reduction by using,
as biocatalysts, both commercial Saccharomyces cerevisiae (Baker’s
yeast) biomass and by nine additional yeast strains, which are
listed in Table 2.
As a general procedure, all the reductions reported in Table 2
were initially performed on a microscale level and the products
Table 2
Relative percentages of the products identified in the crude reaction mixtures after yeast-catalysed reduction of 10a–d
Entry
Yeast strains
Products derived from yeast-catalysed reduction of 10a, 10b, 10c and 10d (rel. %)
10b 10c 10d
14c 17d
60
10a
13a
14a
13b
15b
16b
18b
13c
17c
8c
13d
14d
15d
8d
1
2
3
4
5
6
7
8
9
Candida sake DBVPG 6162
Candida shehatae DBVPG 6850
32
89
92
78
90
91
14
87
82
68
11
8
22
10
9
86
13
18
—
100
30
50
—
—
—
—
—
—
—
—
—
20
—
—
—
—
44
50
—
—
—
—
40
100
—
—
—
—
—
24
—
—
—
—
—
100
—
—
—
—
—
35
—
6
70
50
—
—
40
—
—
40
—
—
—
—
63
25
—
60 (syn)
35 (syn)
—
Debaryomyces nepalensis DBVPG 7123
Hanseniaspora occidentalis DBVPG 6798
Kluyveromyces lodderae DBVPG 6308
Saccharomyces cerevisiae DBVPG 6040
Saccharomyces exiguus DBVPG 6469
Yarrowia lipolytica DBVPG 6629
Yarrowia lipolytica DBVPG 7070
60
100
28
30
37
11
53
24
34
34
—
56
50
100
80
33
—
—
—
20
47
48
30
20
—
—
—
—
—
25
5
—
13
—
8 (anti) 25 (syn)
42 (syn)
16 (syn)
33 (syn)
36 (syn)
70
80
100
100
—
34
—
30
26
20
—
—
—
COOH
Ar
Ar
Ar
Ar
a
O
OH COOH
O
COOEt
O
COOH
O
13a-d
14a,[b],c,d
15b,d
16b
COOEt
COOEt
COOEt
Ar
yeast
Ar
Ar
b
c
O
OH COOH
O
COOEt
O
8c, 8d
( )-10a-d
17c,d
b.y.
N
COOH
18b
COOEt
Ar
(+)-8d, (—)-9d
OH COOEt
11d, 12d
Scheme 2. General pathways of yeast-catalysed transformations.

C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
2029
hemiesters 17c and 17d, these can be easily converted into their
corresponding ethyl -heteroarylparaconates (+)-8c and 8d, both
having a cis-geometry.
allows the isolation of the unreacted lactonic ester, the aqueous
phase is acidified to pH 2, in order to separate the acidic product.
In the present case, acidification was not possible, because of the
presence of the pyridine ring. Therefore, the aqueous phase was
evaporated, and the resulting crude reaction mixture was treated
with trimethylsilyl chloride24 in methanol in order to esterify the
carboxy groups present. When the carboxy group was the result
of ring opening, under the acidic esterification conditions, lactoni-
sation partially occurred in all cases but one, namely in the case of
the 2-pyridyl derivative 8b, for which the hydroxy ethyl methyl
diester 19b was isolated (Fig. 3).
c
Finally, pathway c involves a retro Claisen reaction with the
formation of the heteroaromatic acid and it is followed only by
the 2-pyridyl derivative 10b, which forms picolinic acid 18b.
The detailed results for the transformations of 10a–d are sum-
marised in Table 2 for all yeast strains used. First of all it should
be mentioned that no traces of the starting materials have been
found in the crude reaction mixtures. This is probably due to the
strong hydrolytic and decarboxylative activity of the yeasts.
The best result was obtained with Candida shehatae DBVPG
6850 (entry 2), which chose route b exclusively; the resulting
hydroxy hemiester 17c was obtained with a syn-configuration
and was isolated in 38% yield. Its chemical lactonisation furnished
N
COOEt
(R)
(S)
OH COOMe
the corresponding c-lactone (+)-8c with 99% ee. The same yeast, in
the bioreduction of the 4-pyridyl derivative 10d, led to the syn-
hydroxy hemiester 17d at 60% conversion.
19b
As for the reduction catalysed by S. cerevisiae (Baker’s yeast)
biomass, only the ketodiester 10d gave satisfactory results, leading
to the corresponding hydroxy diesters syn-11d and anti-12d in a
Figure 3. Ethyl methyl hydroxydiester 19b.
The results are summarised in Tables 3 and 4. As can be seen
from Table 3, the cis-
c-lactones 8a–d were hydrolysed regioselec-
7:3 ratio. After lactonisation, the corresponding c-lactones (+)-8d
tively at the lactone group, whereas hydrolyses of the trans-
tones 9a–d (Table 4) were not regioselective, leading to mixtures of
c-lac-
with 99% ee and (ꢀ)-9d with 30% ee were obtained. Baker’s yeast
biomass reduction of 10c led to the isolation of 10% of (+)-8c with
76% ee and 15% of (ꢀ)-9c with 94% ee, although in low yield, since
the main product was the ethyl ketoester22 of 14c (75%). The
remaining substrates 10a and 10b gave, as the only product, the
corresponding ethyl ester23 of 14a (30%) and that22 of 14b (25%),
respectively.
acids.
Since the reactions were stopped at high conversion values,
both the unreacted lactonic esters 8a–d and 9a–d could be isolated
with good enantiomeric excesses, the former ranging from 94% to
99%, and the latter from 80% to 93%. As a consequence, both the
lactonic esters 8a–d and 9a–d (derived from lactonisation of the
ring fission products syn-17a–d and anti-17a–d, respectively),
and the lactonic acids 20a–d (formed by hydrolysis of the ethoxy-
carbonyl group), were obtained with low enantiomeric excesses.
Only in the case of 9a could the lactonic acid trans-(+)-20a and
the lactonic ester (ꢀ)-9a, deriving from hydrolytic opening of the
heterocycle, be easily separated (see Section 4). Interestingly,
although the enantiomeric ratio was low, the two reactions exhib-
ited opposite enantioselectivity: the enantiomer (+)-9a was hydro-
lysed at its ethoxycarbonyl group, whereas (ꢀ)-9a was hydrolysed
at the lactonic group. When the same reaction was performed in a
biphasic system (diisopropyl ether/buffer pH 7.4) hydrolysis took
place at the alkoxycarbonyl group, although very slowly (8 d vs
1.5 h) and with poor selectivity (E = 8).
2.3. Enzymatic hydrolyses of lactones 8a–d and 9a–d
Horse liver acetone powder (HLAP) proved the most efficient for
all the c-substituted-c-paraconic acid esters 8a–d and 9a–d pre-
pared by chemical reduction of ketodiesters 10a–d and subsequent
acidic cyclisations (Scheme 1), since hydrolyses with Porcine Pan-
creatic Lipase (PPL) and a-chymotrypsin (a-CT) were not enantio-
selective. Prior to enzymatic hydrolyses, the amount of chemical
hydrolysis was determined for all substrates. They were hydroly-
sed for about 10% when stirred in 0.1 M phosphate buffer at pH
7.4, for 12 h, giving a mixture of lactonic acid and lactonic ester,
this latter was a consequence of the lactone ring opening and ring
closure, under the reaction workup.
Finally, it should be underlined that enzymatic hydrolyses of
trans-lactones proceeded faster than those of their cis-counterparts
(1–3 h vs 16–39 h, respectively).
The workup of the enzymatic hydrolyses of the
tones deserves a comment. Usually, as in the case of the
yl-substituted
c
-pyridyl lac-
-phen-
c-lactonic ester, after extraction at pH 7.4, which
c
Table 3
Hydrolyses of compounds 8a–d performed with HLAP
COOEt
Ar
unreacted
(+) or (—)-8a-d
HLAP
( )-8a-d
+
OH COOH
(—) or (+)-syn-17a-d
MeOH
Me3SiCl
(—) or (+)-8a-d
Substrate
Ea
Conv.b
Time (h)
Unreacted ester
eec (%)
Lactonic ester from ring fission
eec (%)
8a
8b
8c
8d
10
13
6
64
65
83
68
25
16
21
39
(+)-8a
94
98
99
99
(ꢀ)-8a
53
53
20
46
(ꢀ)-8b
(+)-8c
(+)-8d
(+)-8b
(ꢀ)-8c
(ꢀ)-8d
12
½ee ð1ꢀee Þꢁ
P
S
ln
ln
ee þee
a
E-value was calculated from the formula containing the enantiomeric excesses of both the substrate and the product25 E ¼
.
S
P
S
½ee ð1þee Þꢁ
P
ee þee
b
c
P
S
Calculated.
Determined by chiral HRGC.

2030
C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
Table 4
Hydrolyses of compounds 9a–d performed with HLAPa at 80% conversionb
COOEt
HOOC
Ar
Ar
HLAP
unreacted
(—)-9a-d
+
( )-9a-d
+
O
O
OH COOH
(+) or (—)-20a-d
(—) or (+)-anti-17a-d
MeOH
(—) or (+)-9a-d
Lactonic ester from ring fission
Me3SiCl
Substrate
Time (h)
Unreacted ester
eec (%)
Lactonic acid
eed (%)
Rel. yield
eec (%)
Rel. yield
9a
9b
9c
9d
1.5
2
1
(ꢀ)-9a
93
80
82
89
(+)-20a
32
47
9
74
75
56
40
(ꢀ)-9a
(+)-9b
(+)-9c
(+)-9d
37
23
73
62
26
25
44
60
(ꢀ)-9b
(ꢀ)-9c
(ꢀ)-9d
20be
20ce
20de
3
11
a
E-values could not be determined, as the reactions were not regioselective.
pH STAT value.
Determined by chiral HRGC.
Determined by chiral HRGC, after esterification as methyl esters.
Not isolated.
b
c
d
e
2.4. Assignment of the absolute configuration of lactones 8
and 9
In methanol they all showed a negative Cotton effect for the n?p*
transition at about 210 nm (Fig. 6), as did (ꢀ)-9b. As a conse-
quence, the (2R,3R)-configuration was attributed to all trans-
lactones.
The ethyl
c
-(2-pyridyl)paraconate (ꢀ)-9b with 80% ee was
hydrolysed with 48% hydrobromide so that the corresponding
hydrobromide salt (ꢀ)-22b was separated. Its single crystal X-ray
analysis revealed the (2R,3R)-absolute configuration (Figs. 4 and 5).
1
Ar = 4-Py
(–)-9d
Ar = 3-Py
(–)-9c
HOOC
(R)
0
Δε -1
-2
Ar = 2-Py
(–)-9b
O
(R)
O
N+
H
(—)
—
Br
-22b
Ar = Ph
(–)-9a
Figure 4. Hydrobromide salt (ꢀ)-22b.
-3
210
250
300
350
λ (nm)
Figure 6. CD spectra of the trans-diastereomers (ꢀ)-9a, (ꢀ)-9b, (ꢀ)-9c and (ꢀ)-9d.
To determine the absolute configuration of cis-lactones 8a–d, an
equilibration reaction under basic conditions was performed on
compound (+)-8a, which largely converted into its isomer (ꢀ)-9a.
Since the reaction site was C-3, the absolute configuration of (+)-
8a was assigned as (2R,3S). This attribution was then extended to
the other cis-lactones (ꢀ)-8b, (+)-8c and (+)-8d, whose CD spectra
exhibited the same positive Cotton effect at about 210 nm (Fig. 7).
3. Conclusions
The usual chemoenzymatic methodology was applied to the
synthesis of enantiopure 2-phenyl and 2-(n-pyridyl) diastereo-
meric paraconic esters. This involves the chemical reduction of
suitable b-ketodiesters followed by lactonisation and enzymatic
resolution of the resulting lactonic esters by means of HLAP. A bet-
ter route would be the direct diastereo- and enantioselective enzy-
matic reduction of the parent ketodiesters, followed by cyclisation
of the resulting hydroxyl diester intermediates. With the availabil-
ity of a series of yeasts, this strategy was adopted, with the aim of
finding the most efficient one. This was found to be C. shehatae,
which proved to only be effective on the ketodiester 10c, eventu-
Figure 5. ORTEP diagram of the molecular cation of (ꢀ)-22b.
Since the ee of (ꢀ)-22b was only 80%, the X-ray structural
determination was performed on two different crystals in order
to guarantee the correctness of the assignment. The lactone confor-
mation of the ring was an envelope type, with C(3) being displaced
by 0.42 Å from the coplanar O(1)/C(2)/C(4)/C(5) atoms. The dihe-
dral angle between the pyridine ring and the mean lactone plane
was 58.1(1)°.
The absolute configuration of the other trans-lactones (ꢀ)-9a,
(ꢀ)-9c and (ꢀ)-9d was determined by analysis of their CD spectra.

C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
2031
removed under reduced pressure and the water solution was
extracted with ether. The organic phase was washed with water
containing a few drops of HCl and dried over anhydrous Na2SO4.
Evaporation under reduced pressure afforded almost pure com-
pound 10a in quantitative yield. All spectroscopic data were in
accordance with the literature.18,19 MS, m/z 278 (4, M+Å), 233
(15), 232 (15), 205 (52), 187 (33), 105 (100), 77 (34).
Ketodiesters 10b, 10c and 10d were prepared according to the
literature.12
3
2
Ar = Ph
(+)-8a
Ar = 3-Py
(+)-8c
Ar = 2-Py
(–)-8b
1
Δε
Ar = 4-Py
(+)-8d
0
4.2.2. Diethyl 2-pyridylcarbonylsuccinate 10b12
Yellow oil. IR (neat) 1732, 1716, 1579. 1H NMR (400 MHz,
CDCl3) d 8.68 (1H, d, J = 4.8 Hz, Het H-6), 8.07 (1H, d, J = 7.7 Hz,
Het H-3), 7.85 (1H, dt, J1 = 7.7, J2 = 1.1 Hz, Het H-4), 7.48 (1H, dd,
J1 = 4.8, J2 = 7.7 Hz, Het H-5), 5.30 (1H, dd, part X of an ABX system,
JAX = 5.9, JBX = 8.4 Hz, CHCOOEt), 4.13 (4H, q, J = 7.3 Hz, 2 OCH2CH3),
3.12 (1H, part B of an ABX system, JAB = 16.8, JBX = 8.4 Hz, CH2COO-
Et), 3.00 (1H, part A of an ABX system, JAB = 16.8, JAX = 5.9 Hz,
CH2COOEt), 1.22 (3H, t, J = 7.3 Hz, CH3CH2O), 1.13 (3H, t, J =
7.3 Hz, CH3CH2O). 13C NMR, (100.1 MHz, CDCl3) d 195.7 (s, C@O),
171.2 (s, COO), 169.6 (s, COO), 152.1 (s, Het C-2), 148.9 (d, Het
C-6), 136.9 (d, Het C-4), 127.3 (d, Het C-5), 122.4 (d, Het C-3),
61.3 (t, OCH2CH3), 60.9 (t, OCH2CH3), 48.5 (d, CHCOOEt), 32.9 (t,
CH2COOEt), 14.0 (q, CH3), 13.8 (q, CH3). MS, m/z 279 (7, M+Å), 233
(56), 206 (29), 187 (33), 178 (19), 160 (100), 132 (14), 96 (12),
78 (14).
-1
-2
200
220
240
260
280
300
λ (nm)
Figure 7. CD spectra of the cis-diastereomers (+)-8a, (ꢀ)-8b, (+)-8c and (+)-8d.
ally leading to lactone (+)-8c with high enantiomeric excess.
Although this procedure may look difficult and tedious, the obtain-
ment of the desired product is straightforward.
4. Experimental
4.1. General
4.2.3. Diethyl 3-pyridylcarbonylsuccinate 10c12
IR spectra were recorded on a Jasco FT/IR 200 spectrophotome-
ter. 1H NMR and 13C NMR spectra were run on a Jeol EX-400 spec-
trometer (400 MHz for proton, 100.1 MHz for carbon), and on a Jeol
EX-270 spectrometer (270 MHz for proton, 67.78 MHz for carbon)
using deuteriochloroform as the solvent and tetramethylsilane as
the internal standard. Chemical shifts are expressed in parts per
million (d). Optical rotations were determined on a Perkin Elmer
Model 241 polarimeter. CD spectra were obtained on a Jasco
J-710 spectropolarimeter (0.1 cm cell). GLC analyses were run on
a Carlo Erba GC 8000 instrument and on a Shimadzu GC-14B
instrument, the capillary columns being OV 1701 (25 m ꢂ
0.32 mm) (carrier gas He, 40 kPa, split 1:50, 150 °C for 2 min,
3 °C/min until 200 °C, 200 °C isotherm), a DiMePe b-cyclodextrin
(25 m ꢂ 0.25 mm) (carrier gas He, 110 kPa, split 1:50, 150 °C iso-
Yellow oil. IR (neat) 1730, 1699, 1589. 1H NMR (400 MHz,
CDCl3) d 9.26 (1H, d, J = 1.6 Hz, Het H-2), 8.81 (1H, dd, J1 = 4.6,
J2 = 1.3 Hz, Het H-6), 8.31 (1H, dt, J1 = 1.6, J2 = 8.0 Hz, Het H-4),
7.45 (1H, dd, J1 = 4.6, J2 = 8.0 Hz, Het H-5), 4.82 (1H, dd, part X of
an ABX system, JAX = 5.9, JBX = 8.6 Hz, CHCOOEt), 4.15 (2H, q,
J = 7.2 Hz, OCH2CH3), 4.13 (2H, q, J = 7.2 Hz, OCH2CH3), 3.20 (1H,
part B of an ABX system, JAB = 17.5, JBX = 8.6 Hz, CH2COOEt), 3.03
(1H, part A of an ABX system, JAB = 17.5, JAX = 5.9 Hz, CH2COOEt),
1.23 (3H, t, J = 7.2, CH3CH2O), 1.16 (3H, t, J = 7.2, CH3CH2O). 13C
NMR, (100.1 MHz, CDCl3) d 193.2 (s, C@O), 179.9 (s, COO), 167.8
(s, COO), 153.5 (d, Het C-2), 149.9 (d, Het C-6), 136.1 (d, Het C-4),
131.5 (s, Het C-3), 123.5 (d, Het C-5), 61.9 (t, OCH2CH3), 61.0 (t,
OCH2CH3), 49.5 (d, CHCOOEt), 32.9 (t, CH2COOEt), 13.9 (q, CH3),
13.7 (q, CH3). MS, m/z 280 (4, M+1), 279 (5, M+Å), 250 (31), 235
(83), 206 (100), 160 (34), 106 (84), 78 (63).
therm) and a ChiraldexTM type G-TA, trifluoroacetyl
c-cyclodextrin
(40 m ꢂ 0.25 mm) (carrier gas He, 180 kPa, split 1:100, 150 °C iso-
therm). Enzymatic hydrolyses were performed using a pH-stat
Controller PHM290 Radiometer, Copenhagen. Microwave reactions
were performed in a CEM Discover MW reactor. Mass spectra were
recorded on a ion trap instrument Finningan GCQ (70 eV). TLC’s
were performed on PolygramÒ Sil G/UV254 silica gel pre-coated
plastic sheets (eluent: light petroleum-ethyl acetate). Flash chro-
matography was run on silica gel for flash chromatography
(BDH). Light petroleum refers to the fraction with bp 40–70 °C
and ether to diethyl ether. 1,8-Diazabicyclo[5.4.0]undec-7-ene
(DBU) was purchased from Acros, and ethyl benzoylacetate,
ethyl bromoacetate, diethyl succinate, ethyl 2-pyridylcarboxylate,
ethyl 3-pyridylcarboxylate and ethyl 4-pyridylcarboxylate were
purchased from Sigma–Aldrich.
4.2.4. Diethyl 4-pyridylcarbonylsuccinate 10d12
Yellow oil. IR (neat) 1732, 1701, 1550. 1H NMR (400 MHz,
CDCl3) d 8.84 (2H, d, J = 6.2 Hz, Het H-2 and H-6), 7.81 (2H, d,
J1 = 6.2 Hz, Het H-3 and H-5), 4.80 (1H, dd, part X of an ABX system,
JAX = 5.5, JBX = 8.8 Hz, CHCOOEt), 4.14 (2H, q, J = 7.1 Hz, OCH2CH3),
4.13 (2H, q, J = 7.1 Hz, OCH2CH3), 3.19 (1H, dd, part B of an ABX sys-
tem, JAB = 17.6, JBX = 8.8 Hz, CH2COOEt), 3.02 (1H, dd, part A of an
ABX system, JAB = 17.6, JAX = 5.5 Hz, CH2COOEt), 1.23 (3H, t, J = 7.1
Hz, CH3CH2O), 1.15 (3H, t, J = 7.1 Hz, CH3CH2O). 13C NMR
(100.1 MHz, CDCl3) d 194.1 (s, C@O), 171.0 (s, COO), 167.7 (s,
COO), 150.6 (2d, Het C-2 and C-6), 142.4 (s, Het C-4), 121.6 (2d,
Het C-3 and C-5), 62.1 (t, OCH2CH3), 61.2 (t, OCH2CH3), 49.6 (d,
CHCOOEt), 33.0 (t, CH2COOEt), 14.0 (q, CH3), 13.8 (q, CH3). MS,
m/z 279 (5, M+Å), 233 (28), 206 (100), 187 (16), 177 (13), 160
(28), 152 (22), 106 (76), 78 (53).
4.2. Syntheses of racemic substrates
4.2.1. Diethyl 2-benzoylsuccinate 10a18,19
4.3. Reduction of ketodiester 10a with sodium borohydride
Ethyl benzoylacetate (9.6 g, 50 mmol) and ethyl bromoacetate
(8.5 g, 106 mmol) were slowly added to a solution of EtONa
(52 mmol) in EtOH (1.2 g of Na in 50 mL of EtOH) at 0 °C under stir-
ring. The mixture was stirred overnight under an Ar atmosphere.
Water was added until complete dissolution of NaBr, EtOH was
Sodium borohydride (83 mg, 2.2 mmol) was slowly added to a
solution of ketodiester 10a (1.03 g, 3.7 mmol) in 7 mL of ethanol,
under stirring at 0 °C for 1 h. At the end of the reaction, monitored
by TLC, the solvent was evaporated under reduced pressure, water

2032
C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
was added, extracted with ether and dried over Na2SO4. Evapora-
tion under reduced pressure afforded a mixture of the syn-hydroxy-
ester 11a and lactones 8a and 9a (27%, 60% and 13%, respectively,
determined by 1H NMR analysis of the crude reaction mixture).
Conversion of 11a into the corresponding lactone cis-8a was accom-
plished by refluxing in toluene with p-toluenesulfonic acid as the
catalyst for 2 h. The mixture was extracted with a solution of
NaHCO3 (5%), dried over Na2SO4. Evaporation under reduced pres-
sure afforded compounds 8a and 9a in the ratio of 4:1, respectively,
as determined by 1H NMR analysis. Equilibration of the mixture
with DBU (2.5 equiv of DBU in CH2Cl2, stirred at room temperature
for 24 h) changed the ratio to 1:4, respectively. The diastereomers
8a and 9a were then separated by flash chromatography (eluent:
light petroleum–ethyl acetate, gradient from 9:1 to 4:1).
7.31 (1H, d, J = 7.4 Hz, Het H-3), 7.23 (1H, ddd J1 = 4.8, J2 = 7.4 Hz,
Het H-5), 5.02 (1H, d, J = 4.0 Hz, CHOH), 4.65 (1H, br s, OH), 4.12
(2H, q, J = 7.1 Hz, OCH2CH3), 4.06 (1H, q, J = 7.1 Hz, OCH2CH3),
4.05 (1H, q, J = 7.1 Hz, OCH2CH3), 3.45 (1H, m, CHCOOEt), 2.77
(2H, part AB of an ABX system, JAB = 17.2 Hz, CH2COOEt), 1.24
(3H, t, J = 7.1 Hz, CH3CH2O), 1.10 (3H, t, J = 7.1 Hz, CH3CH2O).
4.4.2. Diethyl (10R*,2R*)-2-[(2-pyridyl)hydroxymethyl]-
butanedioate 12b
The following data were obtained from the 1H NMR spectrum of
the crude reaction mixture: 1H NMR (400 MHz, CDCl3) d 5.25 (1H,
d, J = 3.7 Hz, CHOH), 3.34 (1H, dt, J1 = 4.2, J2 = 9.9 Hz, CHCOOEt),
2.22 (1H, dd, J1 = 4.2, J2 = 17.0 Hz, CH2COOEt).
4.4.3. Ethyl cis-5-oxo-2-(2-pyridyl)tetrahydro-3-
furancarboxylate 8b
4.3.1. Diethyl (10R*,2S*)-2-(hydroxymethylphenyl)butanedioate
11a
Colourless oil, 0.30 g, 30% yield. IR (neat) 1776, 1726, 1595,
The following data were obtained from the 1H NMR spectrum of
the crude reaction mixture: 1H NMR (400 MHz, CDCl3) d 4.92 (1H,
dd, J1 = 5.1, J2 = 7.3 Hz, CHOH), 4.17 (2H, q, J = 7.2 Hz, OCH2CH3),
4.08 (2H, q, J = 7.2 Hz, OCH2CH3), 3.20 (1H, m, CHCOOEt), 3.19
(1H, d, J = 5.1 Hz, OH), 2.57 (1H, dd, part B of an ABX system,
JAB = 16.8, JBX = 8.8 Hz, CH2COOEt), 2.44 (1H, dd, part A of an ABX
system, JAB = 16.8, JAX = 5.1 Hz, CH2COOEt), 1.21 (6H, t, J = 7.2 Hz,
2CH3CH2O).
1579. 1H NMR (400 MHz, CDCl3)
d 8.56 (1H, dd, J1 = 4.4,
J2 = 0.7 Hz, Het H-6), 7.71 (1H, dt, J1 = 7.9, J2 = 1.6 Hz, Het H-4),
7.38 (1H, d, J = 7.9 Hz, Het H-3), 7.25 (1H, ddd, J1 = 4.4, J2 = 7.9,
J3 = 0.9 Hz, Het H-5), 5.76 (1H, d, J = 8.0 Hz, H-2), 3.82 (3H, m,
OCH2CH3 + H-3), 3.24 (1H, dd, J1 = 7.0, J2 = 17.6 Hz, H-4), 2.82 (1H,
dd, J1 = 8.8, J2 = 17.6 Hz, H-4), 0.90 (3H, t, J = 7.1, CH3CH2O). 13C
NMR (100.1 MHz, CDCl3) d 175.1 (s, C-5), 169.6 (s, COOEt), 155.6
(s, Het C-2), 149.3 (d, Het C-6), 136.7 (d, Het C-4), 123.5 (d, Het
C-3), 121.4 (d, Het C-5), 80.9 (d, C-2), 61.1 (t, OCH2CH3), 45.4 (d,
C-3), 31.5 (t, C-4), 13.7 (q, CH3). MS, m/z 235 (15, M+Å), 162 (100),
118 (51), 108 (12), 96 (11). HRGC (OV1701): tR = 19.0 min.
4.3.2. Ethyl cis-5-oxo-2-phenyltetrahydro-3-furancarboxylate
8a20
White solid; mp 74–76 °C, [lit.20a mp 74–75 °C], 0.52 g, 60%
yield. All spectroscopic data are in accordance with the literature.20
13C NMR (100.1 MHz, CDCl3) d 175.1 (s, C-5), 169.6 (s, COOEt),
135.2 (s, Ph), 128.8 (d, Ph), 128.4 (2d, Ph), 125.7 (2d, Ph), 81.2 (d,
C-2), 61.2 (t, OCH2CH3), 46.5 (d, C-3), 31.6 (t, C-4), 13.5 (q, CH3).
HRGC (OV1701): tR = 18.4 min.
4.4.4. Ethyl trans-5-oxo-2-(2-pyridyl)tetrahydro-3-
furancarboxylate 9b
Colourless oil, 0.35 g, 35% yield. IR (neat) 1776, 1726, 1595, 1579.
1H NMR (400 MHz, CDCl3) d 8.63 (1H, dd, J1 = 4.0, J2 = 1.0 Hz, Het H-
6), 7.75 (1H, dt, J1 = 7.7, J2 = 1.8 Hz, Het H-4), 7.43 (1H, d, J = 7.7 Hz,
Het H-3), 7.30 (1H, m, Het H-5), 5.75 (1H, d, J = 5.1 Hz, H-2), 4.25
(1H, q, J = 7.2 Hz, OCH2CH3), 4.24 (1H, q, J = 7.2 Hz, OCH2CH3), 3.80
(1H, dt, J1 = 5.1, J2 = 8.3 Hz, H-3), 2.97 (2H, apparent d, J = 8.3 Hz, part
AB of an ABX system, H-4), 1.29 (3H, t, J = 7.2 Hz, CH3CH2O). 13C NMR
(100.1 MHz, CDCl3) d 174.5 (s, C-5), 171.2 (s, COOEt), 156.2 (s, Het C-
2), 149.7 (d, Het C-6), 136.9 (d, Het C-4), 123.6 (d, Het C-3), 121.4 (d,
Het C-5), 82.0 (d, C-2), 61.7 (t, OCH2CH3), 45.3 (d, C-3), 31.4 (t, C-4),
13.9 (q, CH3). MS, m/z 235 (10, M+Å), 190 (34), 162 (100), 145 (15),
134 (51), 118 (66), 106 (23), 96 (13), 95 (13), 79 (16). HRGC
(OV1701): tR = 18.2 min.
4.3.3. Ethyl trans-5-oxo-2-phenyltetrahydro-3-furan-
carboxylate 9a20
Colourless oil, 0.37 g, 43% yield. All spectroscopic data are in
accordance with the literature.20 13C NMR (100.1 MHz, CDCl3) d
174.2 (s, C-5), 170.7 (s, COOEt), 138.0 (s, Ph), 128.9 (2d, Ph),
128.8 (d, Ph), 125.4 (2d, Ph), 82.2 (d, C-2), 61.9 (t, OCH2CH3), 48.7
(d, C-3), 32.2 (t, C-4), 14.1 (q, CH3). HRGC (OV1701): tR = 17.1 min.
4.4. Reduction of the ketodiester 10b with sodium borohydride
To a solution of 1.20 g (4.3 mmol) of ketodiester 10b in 10 mL of
ethanol, 82 mg of NaBH4 was added. The mixture was stirred for
30 min in an ice bath. At the end of the reaction, water was added
and the solution was evaporated to dryness. The crude reaction
mixture, consisting of the syn- and anti-alcohols 11b (67%) and
12b (20%), respectively, together with lactone 8b (13%), was re-
fluxed in toluene in the presence of p-toluenesulfonic acid (PTSA)
for 15 h. The mixture was extracted with 5% NaHCO3 solution,
and the organic phase was dried over Na2SO4. Evaporation under
reduced pressure afforded compounds 8b and 9b in the ratio of
about 3:1, respectively, determined by 1H NMR analysis. Equilibra-
tion of the mixture with some drops of DBU in refluxing toluene
changed the ratio into 1:4, respectively. The diastereomers 8b
and 9b were separated by flash chromatography (eluent: light
petroleum–ethyl acetate, gradient from 3:1 to 3:2).
4.5. Reduction of the ketodiester 10c with sodium borohydride
The reduction was carried out as indicated for ketodiester 10b.
After the usual workup, the mixture of the syn-alcohol 11c (77%)
and lactones 8c (18%) and 9c (5%) was refluxed in toluene with
p-toluenesulfonic acid for 15h. Compounds 8c and 9c were ob-
tained in the ratio of about 4:1, respectively. Equilibration of the
mixture with DBU (some drops of DBU in toluene refluxed under
stirring for 5 h) changed the ratio to 1:4, respectively. The diaste-
reomers were then separated by flash chromatography (eluent:
light petroleum–ethyl acetate 3:7).
4.5.1. Diethyl (10R*,2S*)-2-[(3-pyridyl)hydroxymethyl]-
butanedioate 11c
The following data were obtained from the 1H NMR spectrum of
the crude reaction mixture: 1H NMR (400 MHz, CDCl3) d 8.54 (2H,
m, Het H-2 and H-6), 7.71 (1H, d, J = 7.8 Hz, Het H-4), 7.30 (1H, m,
Het H-5), 5.03 (1H, d, J = 6.6 Hz, CHOH), 4.16 (2H, q, J = 7.1 Hz,
OCH2CH3), 4.09 (2H, q, J = 7.3 Hz, OCH2CH3), 3.23 (1H, m, CHCOO-
Et), 2.59 (2H, part AB of an ABX system, CH2COOEt), 1.23 (3H, t,
J = 7.1 Hz, CH3CH2O), 1.20 (3H, t, J = 7.3 Hz, CH3CH2O).
4.4.1. Diethyl (10R*,2S*)-2-[(2-pyridyl)hydroxymethyl]-
butanedioate 11b
The following data were obtained from the 1H NMR spectrum of
the crude reaction mixture: 1H NMR (400 MHz, CDCl3) d 8.53 (1H,
d, J = 4.8 Hz, Het H-6), 7.70 (1H, dt, J1 = 7.4, J2 = 1.5 Hz, Het H-4),

C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
2033
4.5.2. Ethyl cis-5-oxo-2-(3-pyridyl)tetrahydro-
3-furancarboxylate 8c
4.6.3. Ethyl cis-5-oxo-2-(4-pyridyl)tetrahydro-3-
furancarboxylate 8d
Colourless oil, 0.40 g, 40% yield. IR (neat): 1788, 1731. 1H NMR
(400 MHz, CDCl3) d 8.59 (2H, m, Het H-2 and H-6), 7.64 (1H, dt,
J1 = 1.7, J2 = 7.9 Hz, Het H-4), 7.32 (1H, dd, J1 = 7.9, J2 = 4.9 Hz, Het
H-5), 5.79 (1H, d, J = 7.7 Hz, H-2), 3.86–3.68 (3H, m, OCH2CH3 +
H-3), 3.09 (1H, dd, J1 = 4.6, J2 = 17.6 Hz, H-4), 2.86 (1H, dd,
J1 = 8.8, J2 = 17.6 Hz, H-4), 0.90 (3H, t, J = 7.1 Hz, CH3CH2O). 13C
NMR (100.1 MHz, CDCl3) d 174.3 (s, C-5), 169.4 (s, COOEt), 150.2
(d, Het C-2), 147.5 (d, Het C-6), 133.3 (d, Het C-4), 131.2 (s, Het
C-3), 123.3 (d, Het C-5), 78.9 (d, C-2), 61.5 (t, CH2O), 46.3
(d, C-3), 31.5 (t, C-4), 13.6 (q, CH3). MS, m/z 236 (7, M+1), 235
(10, M+Å), 191 (100), 178 (14), 161 (30), 149 (30), 134 (30), 118
(23), 108 (57), 106 (31), 80 (18), 78 (17). HRGC (OV1701):
tR = 22.4 min.
Colourless oil, 0.40 g, 40% yield. IR (neat): 1793, 1726. 1H NMR
(400 MHz, CDCl3) d 8.65 (2H, br s, Het H-2 and H-6), 7.27 (2H, br s,
Het H-3 and H-5), 5.71 (1H, d, J = 7.7 Hz, H-2), 3.84–3.70 (3H, m,
OCH2CH3 and H-3), 2.95 (2H, part AB of an ABX system,
JAB = 17.6 Hz, H-4), 0.90 (3H, t, J = 7.2 Hz, CH3CH2O). 13C NMR
(100.1 MHz, CDCl3) d 174.1 (s, COO), 169.2 (s, COOEt), 149.8 (2d,
C-2 and C-6), 144.3 (s, C-4), 120.6 (2d, C-3 and C-5), 79.4 (d,
C-2), 61.6 (t, OCH2CH3), 46.1 (d, C-3), 31.8 (t, C-4), 13.6 (q, CH3).
MS, m/z 235 (37, M+Å), 207 (18), 192 (44), 176 (18), 161 (100),
147 (19), 134 (21), 128 (33), 117 (21), 108 (72), 106 (45), 100
(35), 91 (14), 79 (28). HRGC (OV1701): tR = 22.2 min.
4.6.4. Ethyl trans-5-oxo-2-(4-pyridyl)tetrahydro-3-
furancarboxylate 9d
4.5.3. Ethyl trans-5-oxo-2-(3-pyridyl)tetrahydro-
Colourless oil, 0.50 g, 50% yield. IR (neat): 1792, 1732, 1602. 1
H
3-furancarboxylate 9c
NMR (400 MHz, CDCl3) d 8.67 (2H, br s, Het H-2 and H-6), 7.31 (2H,
d, J = 5.5 Hz, Het H-5 and H-3), 5.66 (1H, d, J = 7.3 Hz, H-2), 4.28
(1H, q, J = 7.0 Hz, OCH2CH3), 4.27 (1H, q, J = 7.0 Hz, OCH2CH3),
3.26 (1H, dt, J1 = 7.3, J2 = 9.0 Hz, H-3), 2.97 (2H, part AB of an ABX
system, JAB = 17.9 Hz, H-4), 1.32 (3H, t, J = 7.0 Hz, CH3CH2O). 13C
NMR (100.1 MHz, CDCl3) d 173.4 (s, COO), 170.2 (s, COOEt), 150.4
(2d, C-2 and C-6), 147.1 (s, C-4), 119.9 (2d, C-3 and C-5), 80.1 (d,
C-2), 62.3 (t, OCH2CH3), 48.1 (d, C-3), 31.8 (t, C-4), 14.1 (q, CH3).
MS, m/z 235 (49, M+Å), 206 (22), 194 (46), 193 (53), 178 (19), 161
(100), 147 (31), 132 (20), 117 (17), 107 (15), 105 (45), 79 (19).
HRGC (OV1701): tR = 21.1 min.
Colourless oil, 0.50 g, 50% yield. IR (neat): 1789, 1731, 1596. 1
H
NMR (400 MHz, CDCl3) d 8.66 (2H, br s, Het H-2 and H-6), 7.73 (1H,
d, J = 7.7 Hz, Het H-4), 7.28 (1H, br s, Het H-5), 5.67 (1H, d,
J = 7.7 Hz, H-2), 4.24 (2H, m, OCH2CH3), 3.34 (1H, dt, J1 = 9.5,
J2 = 7.7 Hz, H-3), 2.97 (2H, part AB of an ABX system, JAB = 17.9 Hz,
H-4), 1.28 (3H, t, J = 7.1 Hz, CH3CH2O). 13C NMR (100.1 MHz, CDCl3)
d 173.5 (s, C-5), 170.1 (s, COOEt), 150.4 (d, Het C-2), 147.4 (d, Het
C-6), 133.7 (s, Het C-3), 133.2 (d, Het C-4), 123.7 (d, Het C-5),
80.0 (d, C-2), 62.2 (t, CH2O), 48.5 (d, C-3), 32.2 (t, C-4), 14.1 (q,
CH3). MS, m/z 236 (14, M+1), 235 (17, M+Å), 193 (100), 191 (51),
177 (26), 161 (50), 147 (25), 134 (32), 121 (20), 108 (33), 106
(57), 91 (14), 89 (15), 78 (27). HRGC (OV1701): tR = 21.2 min.
4.7. General procedure for the reduction of ketodiesters 10a–d
catalysed by Baker’s yeast biomass
4.6. Reduction of the ketodiester 10d with sodium borohydride
Commercial S. cerevisiae (Baker’s yeast) biomass (4 g) in 8 mL of
water was preincubated at 50 °C for 30 min, then the ketodiester
(0.36 mmol) was added and the mixture stirred at room tempera-
ture. The course of the reduction was monitored by HRGC. At the
end of the reaction, 5% NaHCO3 solution was added to deprotonate
the pyridine rings after which the broth was extracted with diethyl
ether. The organic phase was dried and evaporated. When the
resulting hydroxy diesters did not cyclise in the reaction medium,
the crude reaction mixture was refluxed in toluene with p-toluene-
sulfonic acid added and the product was purified by flash
chromatography.
After 16 days and 30% conversion, ketodiester 10a afforded the
corresponding ethyl ester23 of 14a. Similarly, ketodiester 10b affor-
ded, after 16 days and 25% conversion, the corresponding ethyl
ester22 of 14b. Conversely, compound 10c was completely
transformed after 5 days, giving the decarboxylated ethyl ester22
of 14c (75%), and lactones (+)-8c (10%) with 76% ee and (ꢀ)-9c
(15%) with 94% ee, the latter two after lactonisation of the crude
reaction mixture.
Reduction was carried out as indicated for ketodiester 10b.
After the usual workup, the mixture of the syn-alcohol 11d (71%)
and the lactones 8d (22%) and 9d (7%) was obtained. When the
reaction was carried out at room temperature, a mixture of the
syn- and anti-alcohols 11d (65%) and 12d (21%), in admixture with
the lactones 8d (8%) and 9d (6%), was obtained. Lactonisation was
completed by microwave irradiation in toluene for 5 min at 120 °C
with a power setting of 250 W. Under these conditions, the cis- and
trans-lactones 8d and 9d, respectively, were obtained in a 2:3 ratio.
Equilibration of the mixture with DBU (some drops of DBU in tol-
uene, under reflux for 5 h) changed the ratio to 1:4, respectively.
The diastereomers 8d and 9d were then separated by flash chro-
matography (eluent: light petroleum–ethyl acetate 3:7).
4.6.1. Diethyl (10R*,2S*)-2-[(4-pyridyl)hydroxymethyl]-
butanedioate 11d
The following data were obtained from the 1H NMR spectrum of
the crude reaction mixture: 1H NMR (400 MHz, CDCl3) d 8.56 (2H,
d, J = 5.0 Hz, Het H-2 and H-6), 7.28 (2H, d, J = 5.0 Hz, Het H-3 and
H-5), 4.98 (1H, d, J = 5.6 Hz, CHOH), 4.12 (2H, q, J = 7.1 Hz,
OCH2CH3), 4.11 (2H, q, J = 7.1 Hz, OCH2CH3), 3.24 (1H, m, CHCOO-
Et), 2.63 (2H, part AB of an ABX system, CH2COOEt), 1.24 (3H, t,
J = 7.1 Hz, CH2CH3), 1.16 (3H, t, J = 7.1 Hz, CH2CH3). 13C NMR
(100.1 MHz, CDCl3) d 172.9 (s, COOEt), 171.3 (s, COOEt), 150.3 (s,
C-4), 149.8 (2d, C-2 and C-6), 121.2 (2d, C-3 and C-5), 72.6 (d,
CHOH), 61.4 (t, OCH2CH3), 61.0 (t, OCH2CH3), 47.7 (d, C-2), 33.2
(t, C-3), 14.1 (q, OCH2CH3), 13.9 (q, OCH2CH3).
Bioreduction of ketodiester 10d afforded, after 6 days and lac-
tonisation of the crude reaction mixture, lactones (+)-8d (70%)
with 99% ee and (ꢀ)-9d (30%) with 30% ee.
4.8. General procedure for the reduction of the ketodiesters
10a–d catalysed by yeast strains
The following procedure is described in detail for the pyridine
derivatives 10b–d, for which particular attention to the pH values
must be paid, owing to a possible protonation of the nitrogen atom.
The yeast strains Candida shehatae DBVPG 6850, Candida sake
DBVPG 6162, Saccharomyces exiguus DBVPG 6469, Hanseniaspora
occidentalis DBVPG 6798, Kluyveromyces lodderae DBVPG 6308, S.
cerevisiae DBVPG 6040, Debaryomyces nepalensis DBVPG 7123, Yarr-
owia lipolytica DBVPG 6629 and Yarrowia lipolytica DBVPG 7070
4.6.2. Diethyl (10R*,2R*)-2-[(4-pyridyl)hydroxymethyl]-
butanedioate 12d
1H NMR (400 MHz, CDCl3) d 5.22 (1H, d, J = 4.0 Hz, CHOH), 3.20
(1H, m, CHCOOEt), 2.73 (1H, dd, J1 = 9.5, J2 = 16.8 Hz, CH2COOEt),
2.40 (1H, dd, J1 = 4.2, J2 = 16.8 Hz, CH2COOEt).

2034
C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
were used. All strains are conserved in the Industrial Yeast Collec-
tion DBVPG (www.agr.unipg.it/dbvpg). Aliquots (100 mL) of 48 h
yeast cell suspensions (calibrated to A580 = 0.5) were used to inocu-
late 25 mL of minimal medium (yeast extract 0.3%, malt extract
0.3%, peptone 0.5%, glucose 1%). After 48 h of incubation on a rotary
shaker (110 rpm) at 25 °C, the cells were harvested by centrifuga-
tion (4000g), washed, frozen at ꢀ80 °C and lyophilised. A prelimin-
ary screening was performed on 15 mg of the ketodiesters 10a–d
using 400 mg of lyophilised cells suspended in 12.5 mL phosphate
buffer (pH 6.5) and incubated on a rotary shaker (110 rpm), at
25 °C, for 10 days. The reaction mixture was treated with 5% NaH-
CO3 to deprotonate the pyridine ring, and the solution was ex-
tracted with ether, the ether phase was dried with sodium sulfate
and analysed by NMR. The 1H NMR spectrum of the crude mixtures,
obtained from the ethereal extracts, indicated that no ester group
was present and therefore the organic molecules should be present
as salts. The mother liquors were centrifuged and the water phase,
separated from the cells, was evaporated to dryness. The solid ob-
tained was suspended in MeOH (0.4 mL), after which trimethylsilyl
chloride (TMSiCl, 0.2 mL) was added and the solution was stirred
overnight, in order to esterify all the carboxylic groups present.
Methanol was evaporated, after which a solution of 5% NaHCO3
was added, again to deprotonate the pyridine ring, and the residue
was treated with ether, to extract the products in neutral forms. The
crude reaction mixtures were analysed by 1H NMR.
4.9. Bioreduction of the ketodiester 10c catalysed by
C. shehatae DBVPG 6850
Ketodiester 10c (0.2 g, 0.7 mmol) and 5.44 g of lyophilised cells
of C. shehatae DBVPG 6850 (obtained as reported above) suspended
in 170 mL phosphate buffer (pH 6.5) were incubated on a rotary
shaker (110 rpm) at 25 °C for 10 days. After incubation, the mix-
ture was centrifuged. The water separated from the cells was
washed three times with water. Water was cooled at ꢀ80 °C and
lyophilised. To the crude reaction mixture (3.58 g), toluene
(50 mL) and PTSA were added, after which the mixture was re-
fluxed for 10 h, washed with 5% NaHCO3 and dried over Na2SO4.
After elimination of the solvent, the residue (0.063 g) was purified
by flash chromatography and (+)-8c (0.04 g, 0.17 mmol, 24% yield)
with 99% ee was isolated.
4.10. Enzymatic hydrolyses of the cis- and trans-c-phenyl
lactones 8a and 9a
Lactone 8a or 9a (0.160 g, 0.68 mmol) was dissolved in 2 mL of
acetone, after which 0.1 M phosphate buffer at pH 7.4 (18 mL) and
HLAP (0.150 g) were added under stirring. The course of the reac-
tion was monitored with a pH-STAT, with continuous addition of
1.0 N NaOH. At high conversion values, the reaction mixtures were
extracted with ether to separate the unreacted lactone (+)-8a
(0.055 g, 35% yield, 94% ee) or (ꢀ)-9a (0.021 g, 13% yield, 93% ee).
The mother liquors were acidified with 5% HCl to pH 2 and
extracted with ether. From the hydrolysis of 8a, the corresponding
hydroxy half-ester syn-17a (0.098 g, 57% yield, 53% ee) was
obtained. From the hydrolysis of 9a a mixture of lactonic acid
(+)-20a and lactonic ester (ꢀ)-9a, formed by ring fission, was
obtained. This mixture was dissolved in ether, after which the
organic phase was washed with a 5% solution of NaHCO3, evapora-
tion of the ether gave (ꢀ)-9a (0.021 g, 13% yield, 37% ee). The aque-
ous phase was filtered on Celite, acidified with 20% HCl to pH 2 and
extracted with ether. Evaporation of the solvent gave (+)-20a
(0.052 g, 37% yield, 32% ee).
Compounds 13a, 14a and 16b were identified as such, while
13b, 13c, 13d, 14c, 14d, 15b, 15d, 17c, 17d and 18b were identified
as their respective methyl esters. Methyl esters of 13a–d and 17d
were identified by comparison of their 1H NMR spectra with their
corresponding diethyl esters, 1H NMR data of compounds 14a23
and 16b26 were in accordance with the literature.
4.8.1. Methyl ester of 4-oxo-4-(3-pyridyl)butanoic acid 14c
1H NMR (400 MHz, CDCl3) d 9.21 (1H, d, J = 1.8 Hz, Het H-2),
8.80 (1H, dd, J1 = 1.5, J2 = 4.8 Hz, Het H-4), 8.25 (1H, dt, J1 = 2.0,
J2 = 8.1 Hz, Het H-6), 7.43 (1H, dd, J1 = 4.8, J2 = 8.1 Hz, Het H-5),
3.75 (3H, s, OCH3), 3.33 (2H, t, J = 6.6 Hz, CH2), 2.81 (2H, t,
J = 6.6 Hz, CH2).
4.10.1. Ethyl (2R,3S)-(+)-cis-5-oxo-2-phenyltetrahydro-3-
4.8.2. Methyl ester of 4-oxo-4-(4-pyridyl)butanoic acid 14d
1H NMR (400 MHz, CDCl3) d 8.82 (2H, dd, J1 = 1.6, J2 = 4.5 Hz,
Het H-2 + H-6), 7.76 (2H, dd, J1 = 1.8, J2 = 4.5 Hz, Het H-3 + H-5),
3.72 (3H, s, OCH3), 3.31 (2H, t, J = 6.6 Hz, CH2), 2.80 (2H, t,
J = 6.6 Hz, CH2).
furancarboxylate 8a
White solid, mp 102–103 °C, 0.056 g, 35% yield; ½a D25
¼ þ10:0 (c
ꢁ
0.54, CH3OH),
De217 = +2.9 (CH3OH), 94% ee, determined by chiral
HRGC: (b-CDX): tR = 151.3 min for the (ꢀ)-(2S,3R)-enantiomer,
and tR = 158.8 min for (+)-(2R,3S)-enantiomer.
4.8.3. Methyl ester of 4-hydroxy-4-(2-pyridyl)butanoic acid 15b
1H NMR (400 MHz, CDCl3) d 8.54 (1H, d, J = 4.4 Hz, Het H-6),
7.70 (1H, dt, J1 = 7.7, J2 = 1.7 Hz, Het H-4), 7.30 (1H, d, J = 7.7 Hz,
Het H-3), 7.22 (1H, m, Het H-5), 4.80 (1H, dd, J1 = 3.7, J2 = 8.1 Hz,
CHOH), 3.67 (3H, s, OCH3), 2.56 (1H, m, H-2), 2.44 (1H, m, H-2),
2.22 (1H, m, H-3), 1.95 (1H, m, H-3).
4.10.2. (3R,4S)-4-Hydroxy-3-ethoxycarbonyl-4-phenylbutanoic
acid 17a
1H NMR (400 MHz, CDCl3) d 7.34 (5H, m, Ph), 5.0 (2H, vbr s, OH),
4.93 (1H, d, J = 7.3 Hz, CHOH), 4.16 (2H, q, J = 7.1 Hz, OCH2CH3),
3.18 (1H, m, CHCOOEt), 2.54 (2H, part AB of an ABX system,
JAB = 17.2 Hz, CH2COOEt), 1.20 (3H, t, J = 7.1 Hz, CH3CH2O). The hy-
droxy half ester syn-17a rapidly converted into (ꢀ)-8a on standing
in CDCl3, 53% ee by chiral HRGC.
4.8.4. Methyl ester of 4-hydroxy-4-(4-pyridyl)butanoate 15d
1H NMR (400 MHz, CDCl3) d 8.56 (2H, d, J = 6.1 Hz, Het H-2 and
H-6), 7.30 (2H, d, J1 = 6.1 Hz, Het H-3 and H-5), 4.81 (1H, dd,
J1 = 4.2, J2 = 7.7 Hz, CHOH), 3.69 (3H, s, OCH3), 2.48 (2H, dt,
J1 = 4.2, J2 = 6.9 Hz, H-2), 2.05 (2H, m, H-3).
4.10.3. Ethyl (2R,3R)-(ꢀ)-trans-5-oxo-2-phenyltetrahydro-3-
furancarboxylate 9a
Colourless oil, 0.020 g, 13% yield, ½a D25
¼ ꢀ55:3 (c 0.58, CH3OH),
ꢁ
D
e
221 = ꢀ2.7 (CH3OH), 93% ee determined by chiral HRGC:
4.8.5. Methyl ester of (3S,4R)-4-hydroxy-3-ethoxycarbonyl-4-
(3-pyridyl)butanoic acid 17c
(b-CDX): tR = 111.5 min for the (+)-(2S,3S)-enantiomer, and tR =
1H NMR (400 MHz, CDCl3) d 8.54 (2H, m, Het H-2 + H-6), 7.71
(1H, d, J = 7.8 Hz, Het H-4), 7.30 (1H, dd, J1 = 4.6, J2 = 7.8 Hz, Het
H-5), 5.03 (1H, d, J = 6.6 Hz, CHOH), 4.16 (2H, q, J = 7.1 Hz,
OCH2CH3), 3.65 (3H, s, OCH3), 3.23 (1H, m, CHCOOEt), 2.59 (2H,
part AB of an ABX system, CH2COOMe), 1.20 (3H, t, J = 7.1 Hz,
OCH2CH3).
114.7 min for the (ꢀ)-(2R,3R)-enantiomer.
4.10.4. (2S,3S)-(+)-trans-5-Oxo-2-phenyltetrahydro-3-
furancarboxylic acid 20a
White solid, mp 116–119 °C, 0.060 g, 37% yield; IR (Nujol):
3429, 1743. 1H NMR (400 MHz, CDCl3) d 7.39 (5H, m, Ph), 5.71

C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
2035
(1H, d, J = 6.7 Hz, H-2), 4.94 (1H, br s, OH), 3.40 (1H, ddd, J1 = 6.7,
4.11.4. Ethyl (2R,3S)-(+)-cis-5-oxo-2-(4-pyridyl)tetrahydro-3-
J2 = 8.3, J3 = 9.5 Hz, H-3), 3.03 (1H, dd, J1 = 8.3, J2 = 17.9 Hz, H-4),
2.94 (1H, dd, J1 = 9.5, J2 = 17.9 Hz, H-4). 13C NMR (100.1 MHz,
CDCl3) d 175.3 (s, COO), 174.0 (s, COO), 137.7 (s, Ph), 129.0 (2d,
Ph), 125.4 (d, Ph), 125.3 (2d, Ph), 82.0 (d, C-2), 48.2 (d, C-3), 31.9
(t, C-4). MS, m/z 206 (22, M+Å), 164 (65), 160 (100), 145 (25), 132
furancarboxylate 8d
Colourless oil, 0.016 g, 10% yield. ½a D25
ꢁ
¼ þ12:6 (c 0.35, CH3OH),
De210 = +2.0 (CH3OH), 99% ee, determined by chiral HRGC:
(b-CDX): tR = 287.7 min for the (ꢀ)-(2S,3R)-enantiomer, and tR =
319.6 min for the (+)-(2R,3S)-enantiomer.
(29), 115 (26), 105 (62), 79 (36), 77 (36). ½a D25
ꢁ
¼ þ19 (c 0.2,
From the mother liquors, (ꢀ)-8d with 46% ee was obtained, in
admixture with (ꢀ)-9d with 12% ee in about 1:1 ratio, after lacton-
isation under microwave irradiation.
CH3OH), 32% ee. The enantiomeric excess of the acid 20a was
determined on its methyl ester.27,21 Compound (+)-20a (2 mg)
was dissolved in MeOH (1 mL) and trimethylsilyl chloride (TMSiCl,
20 lL) was added, the solution was stirred overnight, the solvent
4.11.5. Ethyl (2R,3R)-(ꢀ)-trans-5-oxo-2-(2-pyridyl)tetrahydro-
evaporated, after which CH2Cl2 (1 mL) was added to the residue.
The solution was filtered and analysed by chiral HRGC: (b-CDX):
tR = 88.2 min for the (+)-(2S,3S) enantiomer, and tR = 91.7 min for
the (ꢀ)-(2R,3R) enantiomer.
3-furancarboxylate 9b
Colourless oil, 0.064 g, 40% yield. ½a D25
¼ ꢀ52:8 (c 0.49, CH3OH),
ꢁ
D
e
213 = ꢀ2.3 (CH3OH). Compound (ꢀ)-9b was obtained with 80%
ee determined by chiral HRGC: (c-CDX): tR = 150.2 min for the
(ꢀ)-(2R,3R)-enantiomer, and tR = 153.2 min for the (+)-(2S,3S)-
4.11. Enzymatic hydrolyses of the cis- and trans-lactones 8b–d
enantiomer.
and 9b–d
From the mother liquors treated as indicated above, a mixture
of (+)-9b with 23% ee and the corresponding methyl ester with
47% ee was obtained.
The appropriate lactone (0.160 g, 0.68 mmol) was dissolved in
2 mL of acetone, 0.1 M phosphate buffer at pH 7.4 (18 mL) and
HLAP (0.150 g) were added under stirring. The course of the reac-
tion was monitored with a pH-STAT, with continuous addition of
1.0 M NaOH. At high conversion values, the reaction mixtures were
extracted with ether to separate the unreacted lactones, namely
(ꢀ)-8b, (+)-8c, (+)-8d and (ꢀ)-9b, (ꢀ)-9c and (ꢀ)-9d, respectively.
The mother liquors were filtered on Celite, evaporated to dryness
and the residue was treated with MeOH (8 mL) and trimethylsilyl
chloride (TMSiCl, 0.4 mL), under stirring overnight. The solvent
was evaporated, a 5% solution of NaHCO3 was added, and the
mother liquors were extracted with ether. The mixture of lactonic
methyl and ethyl esters obtained was analysed by chiral HRCG.
Only in the hydrolysis of 8b the alcoholic precursor 19b could be
fully characterised as the ethyl methyl diester.
4.11.6. Ethyl (2R,3R)-(ꢀ)-trans-5-oxo-2-(3-pyridyl)tetrahydro-
3-furancarboxylate 9c
Colourless oil, 0.038 g, 24% yield. Compound (ꢀ)-9c was obtained
with82%ee. From themotherliquors, a mixtureof(+)-9cwith73%ee
and the corresponding methyl ester with 9% ee was obtained.
Lactonic ester (ꢀ)-9c with 82% ee was submitted to further
enzymatic resolution in order to increase the enantiomeric excess.
½
a 2D5
ꢁ
¼ ꢀ51:6 (c 0.5, CH3OH),
D
e
215 = ꢀ1.0 (CH3OH), 94% ee, deter-
mined by chiral HRGC: (b-CDX): tR = 220.7 min for the (+)-(2S,3S)-
enantiomer, and tR = 226.8 min for the (ꢀ)-(2R,3R)-enantiomer.
4.11.7. Ethyl (2R,3R)-(ꢀ)-trans-5-oxo-2-(4-pyridyl)tetrahydro-
3-furancarboxylate 9d
Colourless oil, 0.044 g, 27% yield. ½a D25
¼ ꢀ48:7 (c 0.23, CH3OH),
ꢁ
4.11.1. Ethyl, methyl (10S,2R)-2-(2-pyridyl)hydro-
xymethylbutandioate 19b
D
e
215 = ꢀ2.2 (CH3OH). Compound (ꢀ)-9d was obtained with 89%
ee, determined by chiral HRGC: (b-CDX): tR = 208.1 min for the
(+)-(2S,3S)-enantiomer, and tR = 216.3 min for the (ꢀ)-(2R,3R)-
enantiomer.
Oil, 0.064 g, 40% yield. IR (Nujol) 3462, 1793, 1726, 1596. 1H
NMR (400 MHz, CDCl3) d 8.54 (1H, d, J = 4.8 Hz, Het H-6), 7.70
(1H, t, J = 7.0 Hz, Het H-4), 7.31 (1H, d, J = 8.0 Hz, Het H-3), 7.24
(1H, m, Het H-5), 5.02 (1H, d, J = 3.7 Hz, CHOH), 4.68 (1H, br s,
OH), 4.05 (2H, q, J = 7.0 Hz, OCH2CH3), 3.67 (3H, s, OCH3), 3.46
(1H, m, CHCOOEt), 2.70 (2H, part AB of an ABX system,
JAB = 17.2 Hz, CH2COOEt), 1.09 (3H, t, J = 7.0 Hz, CH3CH2O). 13C
NMR (100.1 MHz, CDCl3) d 172.5 (s, COO), 172.1 (s, COO), 158.9
(s, Het C-2), 148.1 (d, Het C-6), 136.7 (d, Het C-4), 122.8 (d, Het
C-3), 121.0 (d, Het C-5), 72.9 (d, CHOH), 60.7 (t, OCH2CH3), 51.8
(q, CH3), 48.0 (d, CHCOOEt), 32.5 (t, CH2COOMe), 13.9 (q, CH3).
On standing in CDCl3 solution compound 19b easily converted into
(+)-8b with 53% ee.
From the mother liquors, a mixture of (+)-9d with 62% ee and
the corresponding methyl ester with 11% ee was obtained.
4.12. (2R,3R)-(ꢀ)-trans-5-Oxo-2-(2-pyridyl)tetrahydro-3-
furancarboxylic acid hydrobromide (ꢀ)-22b
To compound (ꢀ)-9b (0.1 g, 0.4 mmol) with 80% ee, water
(2 mL) and 48% HBr (0.150 mL) were added, after which the solu-
tion was refluxed for 30 min and the solvent was evaporated. After
three weeks at room temperature, crystals of pure (ꢀ)-22b were
collected and analysed by X-ray crystallography. The 1H NMR spec-
trum of the crude reaction mixture showed that acidic conditions
completely hydrolysed the ester function and opened the lactone
ring for 20%, giving the corresponding hydroxy diacid which was
identified spectroscopically.
4.11.2. Ethyl (2R,3S)-(ꢀ)-cis-5-oxo-2-(2-pyridyl)tetrahydro-3-
furancarboxylate 8b
Colourless oil, 0.056 g, 35% yield. ½a D25
¼ ꢀ7:3 (c 0.49, CH3OH),
ꢁ
De215 = +2.0 (CH3OH), 98% ee, determined by chiral HRGC:
Compound (ꢀ)-22b: Colourless crystals, mp 149–152 °C,
½
a 2D5
ꢁ
¼ ꢀ23:4 (c 0.47, H2O) for the 4:1 mixture of (ꢀ)-22 and the
(b-CDX): tR = 161.8 min for the (+)-(2S,3R)-enantiomer, and tR =
175.8 min for the (ꢀ)-(2R,3S)-enantiomer.
corresponding hydroxy diacid. 1H NMR (400 MHz, D2O) d 8.59
(1H, d, J = 5.5 Hz, Het H-6), 8.42 (1H, t, J = 8.0 Hz, Het H-4), 7.97
(1H, d, J = 8.0 Hz, Het H-3), 7.85 (1H, t, J = 6.8 Hz, Het H-5), 5.93
(1H, d, J = 8.4 Hz, H-2), 3.64 (1H, q, J = 8.8 Hz, H-3), 2.95 (2H, part
AB of an ABX system, JAB = 17.8 Hz, H-4). 13C NMR (100.1 MHz,
D2O + one drop of CD3OD as the reference) d 177.2 (s, COO),
173.7 (s, COO), 152.0 (s, Het C-2), 147.9 (d, Het C-4), 143.0 (d,
Het C-6), 127.8 (d, Het C-5), 125.8 (d, Het C-3), 78.7 (d, C-2), 47.0
(d, C-3), 31.9 (t, C-4).
4.11.3. Ethyl (2R,3S)-(+)-cis-5-oxo-2-(3-pyridyl)tetrahydro-3-
furancarboxylate 8c
Colourless oil, 0.056 g, 35% yield. ½a D25
¼ þ14:7 (c 0.6, CH3OH),
ꢁ
De209 = +2.6 (CH3OH), 99% ee, determined by chiral HRGC:
(b-CDX): tR = 276.4 min for the (ꢀ)-(2S,3R)-enantiomer, and tR =
291.6 min for the (+)-(2R,3S)-enantiomer.
From the mother liquors (ꢀ)-8c with 20% ee was obtained.

2036
C. Forzato et al. / Tetrahedron: Asymmetry 19 (2008) 2026–2036
4.12.1. (10R,2R)-2-[(2-Pyridyl)hydroxymethyl]butanedioic acid
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1H NMR (400 MHz, D2O) d 8.53 (1H, d, J = 5.8 Hz, Het H-6), 8.42
(1H, t, J = 8.0 Hz, Het H-4), 7.90 (1H, d, J = 8.0 Hz, Het H-3), 7.82
(1H, t, J = 6.9 Hz, Het H-5), 5.49 (1H, d, J = 4.0 Hz, CHOH), 3.27
(1H, m, dt, J1 = 4.7, J2 = 9.4 Hz, CHCOOH), 2.62 (1H, dd, J1 = 9.4,
J2 = 17.2 Hz, CH2COOH), 2.16 (1H, dd, J1 = 4.7, J2 = 17.2 Hz,
CH2COOH). 13C NMR (100.1 MHz, D2O + one drop of CD3OD as the
reference) d 176.1 (s, COO), 175.2 (s, COO), 155.6 (s, Het C-2),
147.9 (d, Het C-4), 141.6 (d, Het C-6), 127.2 (d, Het C-5), 125.8
(d, Het C-3), 69.8 (d, C-2), 48.1 (d, C-3), 30.6 (t, C-4).
4.13. X-ray single crystal analysis of compound 22bꢃH2O
Diffraction data were collected at room temperature on a Non-
ius DIP-1030H system with Mo-K
a radiation (k = 0.71073 Å). Cell
refinement, indexing and scaling of the data sets were carried
28
out using DENZO and SCALEPACK. The structure was solved by direct
methods and Fourier analyses,29 and refined by the full-matrix
least-squares method based on F2.29 All calculations were
performed using the WINGX System, Ver 1.70.01.30
14. Stampfer, W.; Edegger, K.; Kosjek, B.; Faber, K.; Kroutil, W. Adv. Synth. Catal.
2004, 346, 57–62.
15. Uskokovic, M. R.; Lewis, R. L.; Partridge, J. J.; Despreaux, C. W. J. Am. Chem. Soc.
1979, 101, 6742–6743.
4.13.1. Crystal data
C10H12BrNO5; M = 306.12, orthorhombic; space group P212121,
a = 7.390(3), b = 8.928(3), c = 18.503(4) Å, V = 1220.8(7) Å3, Z = 4,
16. (a) Chin-Joe, I.; Nelisse, P. M.; Straathof, A. J. J.; Jongejan, J. A.; Pronk, J. T.;
Heijnen, J. J. Biotechnol. Bioeng. 2000, 69, 370–376; (b) Ushio, K.; Yamauchi, S.;
Masuda, K. Biotechnol. Lett. 1991, 13, 495–500; (c) Glänzer, B. I.; Faber, K.;
Griengl, H.; Roher, M.; Woehrer, W. Enzyme Microb. Technol. 1988, 10, 744–749;
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(e) Iriuchijima, S.; Keiyu, A. Agric. Biol. Chem. 1981, 45, 1389–1399.
17. (a) Perrone, M. G.; Santandrea, E.; Scilimati, A.; Syldatk, C.; Tortorella, V.
Tetrahedron: Asymmetry 2005, 16, 1473–1477; (b) Drioli, S.; Nitti, P.; Pitacco,
G.; Tossut, L.; Valentin, E. Tetrahedron: Asymmetry 1999, 10, 2713–2728; (c)
Brooks, D. W.; Wilson, M.; Webb, M. J. Org. Chem. 1987, 52, 2244–2248.
18. Mattson, A. E.; Bharadwaj, A. R.; Scheidt, K. A. J. Am. Chem. Soc. 2004, 126, 2314–
2315.
19. Metten, B.; Kostermans, M.; Van Baelen, G.; Smet, M.; Dehaen, W. Tetrahedron
2006, 62, 6018–6028.
20. (a) Shimada, S.; Hashimoto, Y.; Sudo, A.; Hasegawa, M.; Saigo, K. J. Org. Chem.
1992, 57, 7126–7133; (b) Fukunishi, K.; Inoue, Y.; Kishimoto, Y.; Mashio, F. J.
Org. Chem. 1975, 40, 628–632.
q
calcd = 1.666 g/cm3, ) = 3.376 mmꢀ1, F(000) = 616. Final
l(Mo-Ka
R = 0.0361, wR2 = 0.0787, S = 0.989 for 163 parameters and 14059
reflections, 2669 unique [Rint = 0.0481], of which 1858 with
I > 2r(I), max positive and negative peaks in DF map 0.425,
ꢀ0.432 e Åꢀ3. Absolute structure parameter 0.028(15).31
Crystallographic data (excluding structure factors) for 22b have
been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication number CCDC 679550. Copies of
the data can be obtained, free of charge, on application to CCDC,
12 Union Road, Cambridge, CB2 1EZ, UK [fax: +44(0)-1223-
336033 or e-mail: deposit@ccdc.cam.ac.uk].
21. Fristad, W. E.; Peterson, J. R. J. Org. Chem. 1985, 50, 10–18.
22. Imoto, H.; Sugiyama, Y.; Kimura, H.; Momose, Y. Chem. Pharm. Bull. 2003, 51,
138–151.
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}
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