Promiscuous substrate binding explains the enzymatic stereoand regiocontrolled synthesis of enantiopure hydroxy ketones and diols

Article abstract of DOI:10.1002/adsc.200900218

Regio- and stereoselective reductions of several diketones to afford enantiopure hydroxy ketones or diols were accomplished using isolated alcohol dehydrogenases (ADHs). Results could be rationalised taking into account different (promiscuous) substrate-binding modes in the active site of the enzyme. Furthermore, interesting natural cyclic diketones were also reduced with high regio- and stereoselectivity. Some of the 1,2 and 1,3-diketones used in this study were reduced by employing a low excess of the hydrogen donor (2-propanol) due to the quasi-irreversibility of these ADH-catalysed processes. Thus, using lower quantities of co-substrate, scale-up could be easily achieved.

Full text of DOI:10.1002/adsc.200900218

FULL PAPERS
DOI: 10.1002/adsc.200900218
Promiscuous Substrate Binding Explains the Enzymatic Stereo-
and Regiocontrolled Synthesis of Enantiopure Hydroxy Ketones
and Diols
Marcela Kurina-Sanz,a,* Fabricio R. Bisogno,b Ivꢀn Lavandera,b
Alejandro A. Orden,a and Vicente Gotorb,*
a
INTEQUI-CONICET, ꢁrea de Quꢂmica Orgꢀnica, Facultad de Quꢂmica, Bioquꢂmica y Farmacia, UNSL, Chacabuco y
Pedernera, 5700 San Luis, Argentina
Fax : (+54)-2652-426-711; e-mail: mkurina@unsl.edu.ar
Departamento de Quꢂmica Orgꢀnica e Inorgꢀnica, Instituto Universitario de Biotecnologꢂa de Asturias, University of
b
Oviedo, 33006 Oviedo, Spain
Fax : (+34)-985-103-448; e-mail: vgs@fq.uniovi.es
Received: April 2, 2009; Revised: May 29, 2009; Published online: July 27, 2009
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.200900218.
Abstract: Regio- and stereoselective reductions of in this study were reduced by employing a low
several diketones to afford enantiopure hydroxy ke- excess of the hydrogen donor (2-propanol) due to
tones or diols were accomplished using isolated alco- the quasi-irreversibility of these ADH-catalysed pro-
hol dehydrogenases (ADHs). Results could be ra- cesses. Thus, using lower quantities of co-substrate,
tionalised taking into account different (promiscu- scale-up could be easily achieved.
ous) substrate-binding modes in the active site of the
enzyme. Furthermore, interesting natural cyclic dike- Keywords: alcohol dehydrogenases; diols; hydrogen
tones were also reduced with high regio- and stereo- transfer; hydroxy ketones; quasi-irreversible reduc-
selectivity. Some of the 1,2- and 1,3-diketones used tion
Introduction
leads to the preparation of anti diols,[5] but the intrin-
sic handicap of the maximum 50% yield still remains.
For decades, synthetic organic chemists have devoted Aldolases have also been employed to afford syn- or
their efforts in developing new and versatile protect- anti-derivatives, although the reversibility of these
ing-deprotecting strategies in order to selectively processes appears as the main drawback.[6]
modify similar reacting moieties in the same mole-
However, the use of alcohol dehydrogenases
cule. Hence, considering the loss of the yields and the (ADHs, also called ketoreductases or carbonyl reduc-
laborious separation techniques in multistep proto- tases) applied to the synthesis of such important com-
cols, a different approach is always desired. Biocataly- pounds has been scarcely developed. In the last few
sis has become a powerful tool for highly stereo- and years, the use of purified or overexpressed ADHs
regioselective transformations due to the intrinsic have gained increasing relevance.[7–14] The require-
chirality of enzymes.[1] In particular, biocatalysed re- ment
of
expensive
nicotinamide
cofactors
gioselective transformations using hydrolases have [NAD(P)H] in those processes has slowed down their
been widely described in the literature,[1,2] while the industrial applicability, although the use of several
use of oxidoreductases has less frequently been per- methodologies to recycle the cofactor have greatly
formed.
improved the efficacy of these biotransformations.[9–18]
It is well established that enantioenriched anti diols In the particular case of the synthesis of enantiopure
can be readily available through epoxide ring-opening hydroxy ketones and/or diols starting from the corre-
starting from the suitable oxirane.[3] On the other sponding diketones employing isolated ADHs, there
hand, syn diols can be obtained via osmium-catalysed are few examples available in the literature.[19–26] In
oxidation of olefins, among other methods.[4] Enantio- other cases plants or micro-organisms are em-
selective epoxide hydrolase-catalysed oxirane opening ployed,[27–32] frequently affording a mixture of stereo-
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Promiscuous Substrate Binding Explains the Enzymatic Stereo- and Regiocontrolled Synthesis
FULL PAPERS
and regioisomers due to the action of several enzymes
with different and/or opposite selectivities. These de-
rivatives are important building blocks of many natu-
ral compounds,[33,34] such as pheromones[35,36] or antitu-
mour agents like discodermolide.[37] As a result, they
are used as chiral precursors for fine chemicals in the
flavour and fragrance, agrochemical, and pharmaceut-
ical industries.[38] a-Hydroxy ketones (also called acy-
loins), constitute well-known derivatives for the syn-
thesis of 1,2-amino alcohols through diastereoselec-
tive reductive amination.[39] Furthermore, short-chain
diols can be employed as starting materials for chiral
polymers[40] or as backbone for chiral ligands applied
to asymmetric transition metal catalysis.[41]
Herein we present a stereo- and regioselective
ADH-catalysed reduction of several diketones of in-
terest (Figure 1) with excellent ees and des, using in-
Figure 1. Diketones employed in this study.
ternal recycling of cofactor (substrate-coupled ap-
proach). We will focus on the different substrate rec- Results and Discussion
ognition modes in order to explain the experimental
results obtained, and we will show for the first time As a first step, several diketones (Figure 1) were
the quasi-irreversible reduction concept[42] applied to chosen as suitable substrates to be reduced by com-
the ADH-catalysed reduction of 1,2- and 1,3-dike- mercial ADHs.[43] Thus, 1,2- (1a–c), 1,3- (1d and e),
tones.
and 1,4-diketones (1f) were firstly used in order to
study the acceptance of the tested enzymes (Scheme 1
and Table 1). Lactobacillus brevis ADH (LBADH),[44]
Scheme 1. Regio- and stereoselective reduction of 1,2-, 1,3-, and 1,4-diketones using ADHs and 2-propanol.
Table 1. ADH-catalysed reduction of diketones 1a–f with 2-propanol.[a]
Entry
Substrate
ADH
2 [%][b]
ee [%][c]
3 [%][b]
ee [%][c]
4 [%][b]
de [%][c]
ee [%][c]
1
2
3
4
5
6
7
8
1a
1a
1a
1b
1b
1b
1c
1c
1c
LBADH
ADH-ꢄAꢅ
ADH-T
LBADH
ADH-ꢄAꢅ
ADH-T
LBADH
ADH-ꢄAꢅ
ADH-T
LBADH
ADH-ꢄAꢅ
LBADH
ADH-ꢄAꢅ
LBADH
ADH-ꢄAꢅ
ADH-T
26
68
3
92
49
3
42
4
–
>97
>97
>97
>97
25
60
62
>99 (R)
>99 (S)
99 (S)
>99 (R)
>99 (S)
99 (S)
>99 (R)
99 (S)
–
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (S)
3
–
6
–
2
3
–
–
–
–
–
–
–
–
–
–
99 (R)
–
66 (R)
–
99 (R)
99 (R)
–
–
–
–
–
–
–
–
–
–
69
32
89
6
46
91
10
49
62
–
–
–
–
–
–
–
91 (2R,3R)
62 (2S,3R)
74 (2S,3R)
99 (2R,3S)
95 (2S,3R)
96 (2S,3R)
99 (2R,3S)
99 (2S,3R)
>99
>99
>99
>99
>99
>99
>99
>99
>99
–
–
–
–
–
–
–
9
99 (2S,3R)
10
11
12
13
14
15
16
1d
1d
1e
–
–
–
–
–
–
–
1e
1f[d]
1f[d]
1f[d]
[a]
Reaction conditions: the corresponding diketone (1a–f, 100 mM) was added to the 50 mM Tris-HCl buffer pH 7.5 with the
corresponding cofactor (1 mM) and 2-propanol (5% v/v).
Measured by GC.
Calculated by chiral GC.
Less than 5% of a by-product was formed.
[b]
[c]
[d]
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Marcela Kurina-Sanz et al.
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alcohol dehydrogenase from Rhodococcus ruber
Looking at these results, it was obvious that the
(ADH-ꢄAꢅ),[45] and ADH from Thermoanaerobacter substrate structure greatly influenced the ADH-cata-
sp. (ADH-T)[46] were selected as biocatalysts to per- lysed reductions. For instance, LBADH mainly pro-
form the corresponding reductions using an excess of vided the unexpected syn diol 4a starting from dike-
2-propanol as hydrogen donor, through a ꢄsubstrate- tone 1a, but when the substrate presented an addi-
coupledꢅ or ꢄbiocatalytic hydrogen transferꢅ ap- tional methylene group in the alkyl chain (1b), hy-
proach.[14] The first enzyme shows a perfect anti- droxy ketone 2b resulted in the main product and
Prelog selectivity, that is, it catalyses the transfer of enantiopure anti diol 4b as minor product. Especially
the hydride to the carbonyl moiety through its si face, the formation of syn-4a was very remarkable since
while the second and third ADHs display an excellent this enzyme has always been described as a perfect
Prelog profile.
anti-Prelog enzyme.[44] These data can be explained
As can be noted, these ADHs accepted all the on the basis of promiscuous substrate binding of 1,2-
tested substrates although showing different behav- diketones at the active site of the enzyme (Scheme 2).
iour. Thus, with diketone 1a, LBADH mainly afford- Thus, these substrates possess two reactive positions,
ed enantiopure syn derivative 2R,3R-4a (entry 1), and therefore different binding modes can be in-
while ADH-ꢄAꢅ mainly provided enantiopure hydroxy volved. For 1,2-diketones such as 1a–c, the carbonyl
ketone S-2a (entry 2) and ADH-T anti diol 2S,3R-4a moiety at position 2 will be reduced following the
(entry 3). For the other 1,2-diketones 1b and 1c, standard fashion, that is, with the methyl group locat-
LBADH showed a high preference for the regio- and ed in the small pocket and the ethylcarbonyl moiety
stereoselective reduction of the carbonyl moiety at in the big one. The conflict arises when the ketone at
position 2, providing R-2b and R-2c and a small quan- position 3 is also reduced. Since location of the nico-
tity of anti diols 2R,3S-4b and 2R,3S-4c (entries 4 and
ACHTUNGTRENtNGNU inamide cofactor is well defined in the active site of
7). Meanwhile, Prelog biocatalysts allowed us to ADHs,[47] the hydride attack will take place through
obtain enantiopure anti diols 2S,3R-4b and 2S,3R-4c the same side, but two possible substrate orientations
(entries 6, 8, and 9). On the other hand, the reduction can exist: i) a similar one as that previously described,
of diketone 1b with ADH-ꢄAꢅ afforded 49% of the locating the hydroxyethyl group into the small bind-
enantiopure hydroxy ketone S-2b (entry 5).
ing site and the ethyl moiety into the bigger site, thus
affording the corresponding anti diols 4a–c
Scheme 2. Promiscuous binding modes of diketones 1a–c and hydroxy ketones 2a–c in the active site of alcohol dehydrogen-
ases: LBADH (binding modes A and B); and ADH-ꢄAꢅ and ADH-T (binding modes C and D).
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Promiscuous Substrate Binding Explains the Enzymatic Stereo- and Regiocontrolled Synthesis
FULL PAPERS
Table 2. ADH-catalysed reduction of natural compounds 1g
and 1h with 2-propanol.[a]
(Scheme 2, binding-modes A and C), or ii) the oppo-
site substrate binding providing the syn derivatives
(Scheme 2, binding modes B and D). Depending on
the pocket sizes of the biocatalyst, one mode will be
favoured at the expense of the second one, and there-
fore different regio- and stereoisomer ratios will be
obtained.
From the obtained results, LBADH favoured the
opposite disposition of hydroxy ketone 2a with re-
gards to diketone 1a (Scheme 2, mode B), while for
alcohols 2b and 2c a similar binding was preferred
(Scheme 2, mode A). For 2b and 2c, since in the re-
verse binding mode a propyl or a butyl moiety, re-
spectively, should be located in the small pocket, this
biocatalyst was not able to afford the corresponding
syn diols. The hydroxyethyl group already seems to
crowd the small pocket of this ADH due to the low
Entry
Substrate
ADH
2 [%][b]
ee [%][c]
1
2
3
4
5
6
1g
1g
1g
1h
1h
1h
LBADH
ADH-ꢄAꢅ
ADH-T
LBADH
ADH-ꢄAꢅ
ADH-T
9
>99 (R)
94 (S)
90 (S)
n. d.
>99 (S)
>99 (S)
90
86
3
95
45
[a]
Reaction conditions: the corresponding diketone (1g and
1h, 50 mM) was added to the 50 mM Tris-HCl buffer
pH 7.5 with the corresponding cofactor (1 mM) and 2-
propanol (5% v/v).
Measured by GC.
Calculated by chiral HPLC. n. d. not determined.
[b]
[c]
conversions obtained (less than 10%) for 4b and 4c gioselectively reduce both substrates on the acetyl
(Table 1, entries 4 and 7). On the other hand, ADH- moiety (Ca) affording highly pure (S)-2g and (S)-2h
ꢄAꢅ and ADH-T always preferred a similar disposition alcohols (entries 2, 3, 5 and 6).
for 1a–c and 2a–c placing the larger acyl chain into
Until this point, we were pleased to find out that
the bigger binding site (Scheme 2, mode C), thus different kinds of diketones could regio- and stereose-
mainly affording the corresponding anti diols 4a–c. lectively be reduced by the tested ADHs. Having in
The larger the difference between the acyl chain and mind the scale-up of these processes, we tried to per-
the hydroxy
favouring the anti derivatives (Table 1, compare en- trations, using less harmful conditions for the biocata-
tries 2–3 with 5–6 and 8–9).
lyst and maximising the atom efficiency[52] of the pro-
ACHTUNGTRENNUNGethyl moiety was, the better were the des form the bioreductions at lower co-substrate concen-
A clearer scenario appeared when 1,3- or 1,4-dike- cess. We envisaged the possible irreversibility in the
tones (1d–f) were subjected to the hydrogen transfer reduction of 1,2- and 1,3-diketones since it has previ-
conditions (Table 1, entries 10–16). ADHs showed ously been described that 1-chloro-2-propanol[42]
perfect regio- and stereoselectivity achieving the re- cannot be oxidised by ADHs due to an intramolecu-
duction of the carbonyl moiety at position 2 to afford lar H-bond interaction between the hydroxy group
enantiopure R-2d–f (in the case of LBADH) or S-2d– and the halogen atom.[53] Thus, 1,2- or 1,3-diketones
f (in the case of ADH-ꢄAꢅ and ADH-T) alcohols. Di- could quasi-irreversibly be reduced since it is well
ketone 1f was less well accepted, obtaining moderate known that the corresponding hydroxy ketones show
(with LBADH) or good conversions (with both a strong intramolecular interaction via H-bonding
Prelog enzymes). In this case another by-product (less network (Figure 2).[54]
than 5%) was formed.
The selective modification of natural compounds in
order to obtain novel derivatives with enhanced bio-
logical activities is a difficult task which organic
chemists must often face.[48,49] In our particular case,
the natural product 6-acetyl-2,2-dimethyl-2,3-dihydro-
4H-chromen-4-one (1g) and the derivative 1-(5-
Figure 2. Intramolecular H-bond interactions in 1,2- and 1,3-
hydroxy ketones.
acetyl-2-methoxyphenyl)-3-methylbut-2-en-1-one (1h)
were also chosen as substrates to study the regio- and
stereoselectivity in the bioreduction processes cata-
lysed by isolated ADHs (Table 2). The former was
In order to demonstrate this fact,[55] we repeated
isolated from Ophryosporus axilliflorus (Griseb.) the LBADH-catalysed reduction of diketones 1b, 1d,
Hieron (Asteraceae). These and other related com- and 1e employing different concentrations of 2-propa-
pounds showed anti-inflammatory activity.[50] Previous nol (from 2 equiv. to 10 equiv.), and results were com-
reductions of diketone 1g using plants were report- pared with that obtained in the case of a non-activat-
ed.[27,51] It remained clear that LBADH was not a suit- ed ketone such as 2-octanone (1i). As can be noticed
able enzyme to perform these bioreductions due to (Table 3), diketones were reduced into the corre-
low conversions (entries 1 and 4). This is in concord- sponding hydroxy ketones (>90%) even at low 2-
ance with the small active site of this biocatalyst. On propanol concentrations (entries 1, 4 and 7). On the
the contrary, ADH-ꢄAꢅ and ADH-T were able to re- contrary, for ketone 1i (entry 10) conversion was 67%
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Marcela Kurina-Sanz et al.
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Table 3. LBADH-catalysed reduction of 1,2- (1b), 1,3-dike-
tones (1d and 1e) and 2-octanone (1i), using different con-
centrations of 2-propanol.[a]
In addition, it has been demonstrated that 1,2- or
1,3-diketones can be reduced via ADH-catalysed hy-
drogen transfer using quasi-irreversible conditions,[42]
that is, employing a low excess of the required co-sub-
strate (2-propanol). This is due to the difficulty of the
enzyme to oxidise backwards the formed hydroxy
ketone due to an intramolecular H-bond interaction
between the hydroxy group and the remaining car-
bonyl moiety. Since a lower amount of the hydrogen
donor (usually used in a huge excess) is needed, the
scale-up of the ADH-catalysed reductions of such
compounds is feasible.
Entry
Substrate
[2-propanol] [M]
2 [%][b]
1
2
3
4
5
6
7
8
9
10
1b
1b
1b
1d
1d
1d
1e
1e
1e
1i
0.2
0.5
1
0.2
0.5
1
0.2
0.5
1
94
95
97
92
91
94
95
94
92
67
0.2
Experimental Section
[a]
Reaction conditions: the corresponding diketone
(100 mM) was added to the 50 mM Tris-HCl buffer
pH 7.5 with the corresponding cofactor (1 mM) and 2-
propanol (200 mM–1 M).
General Remarks
Alcohol dehydrogenases and ketones 1a–1f, 1i were pur-
chased from commercial sources. Ketone 1g was isolated
from aerial parts of Ophryosporus axilliflorus (Griseb.)
Hieron according to Favier et al.[50] The same procedure also
afforded the diketone 1-(5-acetyl-2-hydroxyphenyl)-3-meth-
ylbut-2-en-1-one which was methylated by classical methods
(Me2SO4, K2CO3, under acetone reflux, 2 h) to obtain 1h.
Racemic alcohols 2a–h, 3a–c and 4a–c were purchased or
synthesised by conventional reduction from the correspond-
ing ketones (NaBH4, MeOH, 08C or room temperature).
All other reagents and solvents were of the highest quality
available. 1 unit (U) of ADH reduces 1.0 mM of acetophe-
none to 1-phenylethanol per minute at pH 7.5 and 308C in
the presence of NAD(P)H. Flash chromatography was per-
formed using silica gel 60 (230–400 mesh).
[b]
Measured by GC.
due to the similar equilibrium constants between the
2-octanone/2-octanol and acetone/2-propanol pairs.[56]
Since a small amount of 2-propanol (2 equiv.) can
be employed to quantitatively reduce this type of sub-
strates, enantiopure (R)-alcohols 2b, 2d, and 2e were
prepared on a higher scale starting from 25 mg of the
corresponding diketones with isolated yields higher
than 80%.
Conclusions
General Protocol for the Biocatalytic Reduction of
Diketones Employing ADHs and 2-Propanol
The bioreduction of diketones has not been extensive-
ly studied,[19–26] and in several cases micro-organisms
or plants[27–32] were used for this purpose. Thus, the
obtained results in terms of regio- and stereoselectiv-
ity usually remained unclear. Herein we have shown
the bioreduction of several diketones of interest cata-
lysed by isolated alcohol dehydrogenases. With the
advantage of using a single enzyme, we have pro-
posed that in reductions of 1,2-diketones, these sub-
strates can be suited within the active site of the
enzyme in two different orientations depending on
the size of the enzyme pockets. Hence, just by choos-
ing the proper catalyst, syn or anti products could be
obtained. For instance, due to its small binding site,
LBADH presents a promiscuous binding for the dike-
tone 1a and the hydroxy ketone intermediate to
afford the corresponding syn-diol. On the other hand,
ADH-ꢄAꢅ and ADH-T favour a similar recognition
mode for both species, thus mainly providing the anti-
derivatives. This is an elegant example of enzyme-
based stereocontrol.[57] In the case of the other dike-
tones studied, perfect regio- and stereoselectivity was
achieved obtaining the corresponding enantiopure hy-
droxy ketones.
In a 1.5-mL Eppendorf vial, 3 U of ADH (Lactobacillus
brevis ADH, Rhodococcus ruber ADH-ꢄAꢅ, or Thermo-
AHCTUNGTREGaNNUN naerobacter sp. ADH) in Tris-HCl buffer [50 mM, pH 7.5,
1 mM NAD(P)H, 1 mM MgCl2 for LB-ADH] were mixed
with 2-propanol (32 mL, 5% v/v) and the corresponding di-
ketone (1a–1f, and 1i, 100 mm; 1g, 1h, 50 mM). Reactions
were shaken at 308C and 120 rpm for 24 h and stopped by
extraction with ethyl acetate (2ꢆ0.5 mL). The organic layer
was separated by centrifugation (2 min, 13000 rpm) and
dried (Na2SO4). Conversions and enantiomeric excesses of
the corresponding alcohols were determined by GC or
HPLC analysis on an achiral or a chiral stationary phase, re-
spectively (see Supporting Information).
General Protocol for the Biocatalytic Reduction of
Diketones Employing LBADH with Different
Concentrations of 2-Propanol
In a 1.5-mL Eppendorf vial, 3 U of LBADH in Tris-HCl
buffer (50 mM, pH 7.5, 1 mM NADPH, 1 mM MgCl2) were
mixed with 2-propanol (200 mM–1 M) and the correspond-
ing diketone (1b, 1d, 1e or 1i, 100 mM). Reactions were
shaken at 308C and 120 rpm for 24 h and stopped by extrac-
tion with ethyl acetate (2ꢆ0.5 mL). The organic layer was
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Promiscuous Substrate Binding Explains the Enzymatic Stereo- and Regiocontrolled Synthesis
FULL PAPERS
separated by centrifugation (2 min, 13000 rpm) and dried
(Na2SO4). Conversions were determined by GC analysis on
an achiral stationary phase (see Supporting Information).
[6] W.-D. Fessner, in: Asymmetric Organic Synthesis with
Enzymes, (Eds.: V. Gotor, I. Alfonso, E. Garcꢂa-Ur-
diales), Wiley-VCH, Weinheim, 2008, pp 275–318.
[7] H. Li, D. Zhu, L. Hua, E. R. Biehl, Adv. Synth. Catal.
2009, 351, 583–588.
[8] M. J. Sorgedrager, F. van Rantwijk, G. W. Huisman,
R. A. Sheldon, Adv. Synth. Catal. 2008, 350, 2322–
2328.
[9] S. Buchholz, H. Grçger, in: Biocatalysis in the Pharma-
ceutical and Biotechnology Industry, (Ed.: R. N. Patel),
CRC Press, Boca Raton, 2007, pp 757–790.
[10] S. M. A. de Wildeman, T. Sonke, H. E. Schoemaker, O.
May, Acc. Chem. Res. 2007, 40, 1260–1266.
[11] J. C. Moore, D. J. Pollard, B. Kosjek, P. N. Devine, Acc.
Chem. Res. 2007, 40, 1412–1419.
Representative Example for Biocatalytic Reduction
of a Diketone: Preparation of (R)-2d
In a 5-mL screw-capped tube 5 U of LBADH in Tris-HCl
buffer (1.8 mL, 50 mM, pH 7.5, 1 mM NADPH, 1 mM
MgCl2) were mixed with 2-propanol (27.5 mL, 0.36 mmol)
and 1d (28 mL, 0.18 mmol). Reactions were shaken at 308C
and 120 rpm for 24 h and stopped by extraction with ethyl
acetate (3ꢆ1 mL). The organic layer was separated by cen-
trifugation (2 min, 13000 rpm) and dried (Na2SO4). Alcohol
2d was purified by flash chromatography (petroleum ether:
EtOAc 4:1) obtaining thus enantiopure (R)-2d as a colour-
less oil; yield: 21.5 mg (83%); [a]2D0: ꢀ13.7 (c 1.5, CHCl3);
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3
1H NMR (CDCl3, 300 MHz): d=0.92 (d, 6H, H-7, JH,H
=
3
6.6 Hz), 1.18 (d, 3H, H-1, JH,H =6.4 Hz), 2.07–2.20 (m, 1H,
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2
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3
17.8, JH,H =3.1 Hz), 4.22 (m, 1H, H-2); 13C NMR (CDCl3,
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(APCI+): m/z (%)=144 (3) [M]+, 129 (6) [MꢀCH3]+, 126
(8) [MꢀH2O]+, 111 (22), 87 (42), 69 (61), 57 (80), and 43
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Acknowledgements
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M.K.-S. is member of the Research Career of CONICET.
A. A.O. is a postdoctoral CONICET fellow. F.R.B. is sup-
ported by the Programme Alban, the European Union Pro-
gram of High Level Scholarships for Latin America (scholar-
ship No. E07D402519AR). I.L. thanks Principado de Astu-
rias for personal funding (Clarꢀn Program). Financial sup-
port from the Spanish Ministerio de Ciencia e Innovaciꢁn
(MICINN, Project CTQ2007-61126) and the Spanish Minis-
terio de Asuntos Exteriores y de Cooperaciꢁn (Programa de
Cooperaciꢁn Interuniversitaria, PCI Iberoamꢂrica MAEC-
AECID, Project A/8856/07) is gratefully acknowledged.
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ꢃ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2009, 351, 1842 – 1848