Aza and oxo Diels-Alder reactions using cis-cyclohexadienediols of microbial origin: Chemoenzymatic preparation of synthetically valuable heterocyclic scaffolds

Article abstract of DOI:10.1016/j.tetasy.2015.10.015

Aza and oxo Diels-Alder reactions using enantiopure cis-cyclohexadienediols were studied. These dienediols were obtained from the biotransformation of monosubstituted arenes using bacterial dioxygenases (toluene and benzoate dioxygenases). Ethyl glyoxylate and its N-tosyl imine were used as dienophiles to afford the corresponding hetero Diels-Alder bicyclic adducts with excellent regio- and stereoselectivities. Quantum chemical calculations at the B3LYP/6-31+G(d,p) level of theory were performed to rationalize the observed selectivities especially the stereochemical aspects of the cycloadditions. The synthetic importance of these adducts is highlighted for the preparation of enantiopure 2,2,3,4,5,6-hexasubstituted piperidine and tetrahydropyran from toluene.

Full text of DOI:10.1016/j.tetasy.2015.10.015

Tetrahedron: Asymmetry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Aza and oxo Diels–Alder reactions using cis-cyclohexadienediols of
microbial origin: chemoenzymatic preparation of synthetically
valuable heterocyclic scaffolds
Mariana Pazos a, Sebastián Martínez b, María Agustina Vila a, Paola Rodríguez a, Nicolás Veiga b,
Gustavo Seoane a, Ignacio Carrera a,
⇑
a Laboratorio de Química Orgánica, Departamento de Química Orgánica, Facultad de Química—Universidad de la República, General Flores 2124, 11800 Montevideo, Uruguay
b Cátedra de Química Inorgánica, Departamento Estrella Campos, Facultad de Química—Universidad de la República, General Flores 2124, 11800 Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
Aza and oxo Diels–Alder reactions using enantiopure cis-cyclohexadienediols were studied. These diene-
diols were obtained from the biotransformation of monosubstituted arenes using bacterial dioxygenases
(toluene and benzoate dioxygenases). Ethyl glyoxylate and its N-tosyl imine were used as dienophiles to
afford the corresponding hetero Diels–Alder bicyclic adducts with excellent regio- and stereoselectivities.
Quantum chemical calculations at the B3LYP/6-31+G(d,p) level of theory were performed to rationalize
the observed selectivities especially the stereochemical aspects of the cycloadditions. The synthetic
importance of these adducts is highlighted for the preparation of enantiopure 2,2,3,4,5,6-hexasubstituted
piperidine and tetrahydropyran from toluene.
Received 6 October 2015
Accepted 21 October 2015
Available online xxxx
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
These systems have been extensively used as the 4p-compo-
nent in diastereofacially selective Diels–Alder cycloadditions to
achieve the preparation of several substituted bicyclo[2.2.2]octe-
nes (Scheme 2). Many examples have been reported for this
type of intermolecular cycloadditions using diols of type 1 and
several dienophiles such as: triazolinediones,5 acetylenes,5b,6
N-substituted maleimides,6,7 maleic anhydride,7b,8 benzyne,9
benzo- and naphthoquinones,9 nitrosyl derivatives,5b,6,10 singlet
Enantiopure cis-cyclohexadienediols have been extensively
used as starting materials for the enantioselective organic
synthesis of complex natural products.1 These compounds can
be prepared from the corresponding arenes by enzymatic
dihydroxylation using bacterial dioxygenases since no other effi-
cient chemical method for their production at a preparative scale
is known.2 The biotransformation is carried out using whole cells
as the biocatalyst to overcome the need of recycling co-factors as
well as to avoid the difficult procedures of enzyme isolation and
oxygen,11
a
-chloroacrylonitriles,12 and cyclopentenones.13 The
exploitation of diols of type 2 as dienes for these cycloadditions
is found in the literature with nitrosyl groups,4c,j,14 alkenes,4f,h
singlet oxygen,4a,15 and urazole dienophiles.4d,j
purification.1f The most commonly used biocatalyst on
a
preparative scale for the production of cis-cyclohexadienediols of
type 1 is the recombinant Escherichia coli JM109 (pDTG601),
which expresses the toluene dioxygenase enzymatic complex
(Scheme 1).3 For the production of cis-cyclohexadienediols of type
2 the microorganism Ralstonia eutropha B9, that expresses a ben-
zoate dioxygenase, has recently been used for organic synthesis
applications.4 The useful array of functional groups present in 1
and 2 allows the use of regio- and stereocontrolled organic trans-
formations resulting in short and efficient synthetic schemes.1f,4a
Herein we have studied the aza and oxo Diels–Alder cycloaddi-
tions of isopropylidene protected cis-cyclohexadienediols using
ethyl glyoxylate and its N-tosyl imine as dienophiles. These dieno-
philes have been used for heterocycle preparation through a hetero
Diels–Alder methodology,16 but to our knowledge, there is no pre-
vious report of their use with cis-cyclohexadienediols to produce
the expected bicyclo[2.2.2]octenes. These heterocyclic scaffolds
present a high synthetic potential for natural product synthesis,
especially for the preparation of enantiopure polysubstituted
piperidines and tetrahydropyrans. Polysubstituted piperidine and
tetrahydropyran heterocycles are important structural motifs
found in a wide range of biologically active natural products, and
are often key pharmacophores in drug design.17 Previous reports
⇑
Corresponding author. Tel.: +598 29247881.
E-mail address: icarrera@fq.edu.uy (I. Carrera).
http://dx.doi.org/10.1016/j.tetasy.2015.10.015
0957-4166/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pazos, M.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.10.015

2
M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
X
X
yields using previously reported standard conditions and were
OH
OH
subjected to hetero Diels–Alder cycloadditions with ethyl
glyoxylate or its N-tosylimine. cis-Cyclohexadienediols of type 2
were prepared by biotransformation of sodium benzoate by
Ralstonia eutropha B9 using a modification of the procedure
published by Hudlicky et al. (Scheme 3b).4b The protection of 2
using dimethoxypropane for the diol moiety and diazomethane
for carboxylic acid esterification afforded compound 4, which
was subjected to the mentioned hetero Diels–Alder cycloadditions.
Ethyl glyoxylate N-tosyl imine 5 was prepared according to the
procedure described by Le Goffic et al. using ethyl glyoxylate and
p-toluenesulphonylisothiocyanate, and used in situ without fur-
ther purification (see Section 4).19 A first attempt to perform the
imino Diels–Alder reaction on substrate 1a afforded the desired
isoquinuclidines 6, as a 75:25 mixture of diastereomers in 23%
yield (Scheme 4a). Structural analysis by NMR showed complete
regioselectivity for the process, which is in agreement with the
charge distribution and electronic features of the reactants (see
computational results below). The facial selectivity for the dieno-
phile addition, exclusively on the less hindered face of the diene
(anti to the bulky isopropylidene group), was proposed according
to previous reports of Diels–Alder reactions on cis-cyclohexadiene-
diols. The major diastereomer was identified as the exo car-
boxyethyl adduct by NOE experiments on 6 (Scheme 4b). The
preference for the exo addition product in Diels–Alder reactions
using imines containing both N-sulfonyl and C-acyl groups is
already well known.16a,20 The whole exo stereochemistry
assignment for 6 was further confirmed by the transformation of
E.coli JM109 (pDTG601)
(toluene dioxygenase)
1,
2,
99 % ee
CO2Na
CO2Na
OH
Ralstonia eutropha B9
(benzoate dioxygenase)
OH
99 % ee
Scheme 1. Production of cis-cyclohexadiendiols of type 1 and 2 using bacterial
dioxygenases.
have used cis-cyclohexadienediols as precursors of polysubstituted
heterocycles using different synthetic methodologies resulting in
multi step procedures.4b,18 Using the above mentioned
methodology we herein report a chemoenzymatic procedure to
transform toluene into an enantiopure polysubstituted piperidine
and tetrahydropyran in a rapid and efficient fashion.
2. Results and discussion
2.1. Experimental results
cis-Cyclohexadienediols of type 1 were produced by the bio-
transformation of monosubstituted arenes with the recombinant
microorganism E. coli JM109 (pDTG601) using a slight modification
of a previous bi-phasic procedure published by our group (see
Section 4).3b Biotransformation yields in g/L of culture broth are
shown in Scheme 3a for each substrate used. Dienediols of type 1
were protected as the isopropylidene ketals 3 with excellent
adduct
6
into the corresponding piperidine followed by
J-coupling analysis (vide infra, Scheme 7).
In addition to the described isoquinuclidines, the oxygenated
bicyclo[2.2.2]octane 7 was obtained in 11% yield as a 91:9
R
N
O
O
Z
H
H
O
X
O
O
O
O
O
O
X
O
Z
X = Cl, Br,
CO2Et
X
O
R-N=O
O
Z = O, NPh, NEt
R = Ac, CO2Bn,
Ph
O
1O2
X = H, Cl, Me
NC
X = Me, H, CF3
MeO2C
MeO2C
Cl
O
X
Cl
OPG
OPG
O
MeO2C
CO2Me
CN
O
O
Me
X
O
1
H
O
X = Me
Ar
X = CF3
N
N
NPh
O
O
H
H
O
O
PhN
O
N
OH
OH
N
O
X = Me
O
O
X
X = Me
O
H
H
X = H, Cl, Me, CF3
O
O
Me
O
Scheme 2. Intermolecular [4 + 2] cycloadditions in cis-cyclohexadienediols of type 1 using different dienophiles. PG = protecting group.
Please cite this article in press as: Pazos, M.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.10.015

M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
3
X
X
X
OH
p-TsOH cat.
O
O
E.coli JM109 (pDTG601)
(a)
DMP - acetone
0 ºC to r.t.
bi-phasic procedure
OH
1, e.e.> 99%
3
1a, X = Me , 23 g/L
1b
, X = Pr , 6 g/L
1c, X = CH2OAc, 7 g/L
1d
, X = Br, 40 g/L
1e, X = CN, 14 g/L
CO2H
CO2Me
O
CO2Na/K
OH
1. TFA (6.0 equiv)
0 ºC to r.t.; 24 hours
Ralstonia eutropha B9
(b)
2. CH2N2
O
OH
50%, 4,
2
, e.e.> 99%
30 g/L
Scheme 3. (a) Arenes biotransformation using E. coli JM109 (pDTG601) in a bi-phasic procedure and the preparation of isopropylidene derivatives 3. (b) Biotransformation of
benzoic acid using Ralstonia eutropha B9 and the preparation of protected diol 4.
Ts
H
CO2Et
O
EtO2C
CH3
N
Ts
5
N
O
O
O
CO2Et
(a)
Toluene, 100 ºC
O
H3C
H3C
1a
O
O
23 %, 6
7:3 exo:endo
7
11 %,
9:91 exo:endo
H
CO2Et
O
nOe
H
CO2Et
O
H
EtO2C
nOe
nOe
O
H
TsN
O
H
H
(b)
H
O
H
H3C
H3C
H3C
O
O
O
7
7
exo
endo
6
exo
Scheme 4. (a) Imino Diels–Alder reaction of ethyl glyoxylate N-tosyl imine 5 and diene 1a. (b) Diastereomer assignment by key NOE correlations.
endo/exo mixture of epimers. These products could arise from the
reaction of diene 1a with ethyl glyoxylate present in the reaction
media as a decomposition product of the N-tosylimine 5 or as
residual starting material of its formation. Key NOE correlations
(Scheme 4a) allowed the stereochemical assignment of the endo
and exo isomers. In addition, the NOE correlation found for endo-
7 shows that the addition of the dienophile occurred anti to the
bulky isopropylidene group. This could be extrapolated for the
exo isomer, which showed a similar J coupling pattern in the 1H
NMR spectra (see compound characterization in Section 4). In this
case, the endo isomer was the major product of the reaction as
expected for this type of cycloaddition.
In order to obtain a useful reaction for synthetic purposes, the
optimization of the formation of isoquinuclidine 6 was carried out
(Table 1). A screening of solvents (Table 1, entries 1–5) revealed
that toluene was the best choice to obtain isoquinuclidines 6 with
lower amounts of bicycles 7. Different concentrations and imine
equivalents were tested and the best conditions found were 0.5 M
1a in toluene and 1.5 equivalents of N-tosylimine 5 at reflux, to pro-
duce isoquinuclidines 6 in 66% yield (Table 1, entry 7). Lewis acid
catalysis [BF3ÁOEt2 and Yb(OTf)3] was not successful since aromati-
zation of the starting cis-cyclohexadienediol took place.
transformations (Scheme 5). In the case of the oxo Diels–Alder reac-
tion, we found that when R = Me, an excellent yield of 88% for 7 was
obtained, which decreased as the size of the alkyl R substituent
increased (Scheme 5a). This could be explained by the increasing
steric hindrance in the diene that precludes the dienophile access.
Regarding the stereoselectivity of the cycloaddition, the exo isomer
was only found in trace amounts for compound 7, while bicycles 8
and 9 were characterized as a single endo diastereomer. For the aza
Diels–Alder reaction, the same trend in the reactions yields for alkyl
R substituents was found, although the values were significantly
lower in all cases. According to these results it seems that cis-cyclo-
hexadienediols are less reactive toward imine 5 than to ethyl
glyoxylate for the studied cycloadditions. Thus, the only substrate
useful for the preparation of the desired isoquinuclidines for
synthetic purposes was found to be the cis-cyclohexadienediol 1a.
In all the aforementioned cases for the aza Diels–Alder reactions,
exo/endo mixtures of diastereomers were found (Scheme 5a).
cis-Cyclohexadienediols with bromo and cyano groups were
tested as electron withdrawing substituents for the diene system
(Scheme 5b). In both cases the only products obtained were 12
and 13, which corresponds to the already well known Diels–Alder
dimerization for these systems.21 This shows that cis-cyclohexadi-
enediols with electron withdrawing groups work better as dieno-
philes than ethyl glyoxylate or its N-tosyl imine under these
conditions.
The optimized conditions were used to study the reaction scope
for the Diels–Alder reaction using N-tosylimine 5 and ethyl glyoxy-
late to produce the already mentioned oxygenated derivatives on
different cis-cyclohexadienediols. For diols of type 1, R groups
with different sizes and electronic content were tested for both
We next studied these reactions on substrate 4 where the diene
has no R substituents and the asymmetry of the system is given by
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4
M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
Table 1
Imino Diels–Alder optimization of diene 1a
Entry
Solvent
1a (mol/L)
Lewis Acid
Imine 5 (equiv)
Time
Temp (°C)
6 (%), exo/endo
7 (%)
1
2
3
4
5
6
7
8
Toluene
MeCN
DCE
0.10
0.10
0.10
0.10
0.10
0.25
0.50
0.25
0.10
0.10
0.10
0.10
—
—
—
—
—
—
—
—
1.5
1.5
1.5
1.5
1.5
1.5
1.5
3.0
1.5
1.5
1.5
1.5
8
8
8
8
8
4
2
2
48
1
100a
100a
23, 75:25
17, 66:34
26, 74:16
—
Aromatization products
50, 75:25
66, 75:25
51, 75:25
No reaction
11
15
16
36
100a
THF
100a
MeOH
Toluene
Toluene
Toluene
Toluene
Toluene
CH2Cl2
Toluene
100a
110
110
110
T.A.
13
15
20
9
—
10
11
1
BF3-OEt2
BF3-OEt2
Yb(OTf)3
À78 °C–rt
À78 °C–rt
À78 °C–rt
Aromatization products
Aromatization products
Aromatization products
1
1
Yields were determined by NMR using 1,1,2-trichloroethylene as internal standard.
a
Reaction run in sealed vial.
a quaternary center supporting the ester and one of the oxygen
atoms of the diol moiety. For the oxo Diels–Alder reaction, harsher
conditions with a higher temperature were required to ensure
complete conversion of the reaction. Compound 14 was the only
product isolated with very good yield (Scheme 6a). NMR analysis
showed complete regio- and stereoselectivity for the process,
obtaining only one diastereomer, anti-endo, which was identified
by NOE analysis (Scheme 6b). This selectivity is remarkable since
there is no evident polarization in the diene moiety that could
account for the observed regioselectivity. In addition, previous
reports of [4 + 2] cycloadditions under these systems have shown
mixture of regioisomers when nitrosyl groups were used as
dienophiles.4c,4j,14
Regarding the aza Diels–Alder reaction on substrate 4, no
expected isoquinuclidines were found, and only trace amounts of
product 14 were isolated (probably because of decomposition of
dienophile 5). Again, N-tosylimine 5 showed less reactivity as a
dienophile to these diene systems than ethyl glyoxylate.
Bicycles endo-6 and endo-7 were subjected to reductive ozonol-
ysis conditions to afford the corresponding piperidine and tetrahy-
dropyran (Scheme 7a) in good yields. J-coupling analysis22 of
compound 15 confirmed the stereochemical assignment (Sche-
me 7b), which is in agreement with the initially proposed structure
of 6 (where the anti-correlation between the nitrogenated bridge
and the isopropylidenedioxy group was proposed according to pre-
vious reports). It is noticeable that the ester present in 6 was con-
served during the reductive conditions to 15, while the same
functionality in endo-7 was reduced to a primary alcohol in 16
probably due to the presence of an alpha oxygen which facilitates
the reduction process.23
In this manner, toluene can be converted into an enantiomeri-
cally pure polysubstituted piperidine or tetrahydropyrane contain-
ing five stereogenic centers in only four synthetic steps. This
highlights the synthetic importance of the prepared bicyclo[2.2.2]
octenes. cis-Cyclohexadienediols have been previously used to
access polysubstituted heterocycles using different synthetic
methodologies.4b,18 The present study complements these
previous reports on the use of enantiopure dienediols as starting
materials for the preparation of the mentioned heterocycles.
2.2. Computational studies
In order to rationalize the regio- and stereoselectivity patterns
for both types of Diels–Alder reactions using cis-cyclohexadienedi-
ols, we performed some theoretical calculations. High level ab initio
methods have proved to give excellent results for energy barrier
estimation of cycloaddition reactions.24 Besides, these electronic
structure calculations have been previously used to predict stere-
ospecificities and to understand the mechanism of Diels–Alder
reactions.25 Therefore, we performed quantum chemical calcula-
tions on some of the cycloaddition reactions at the B3LYP/6-31+G
(d,p) level of theory. This allowed us to thermodynamically and
structurally characterize the transition states and related minima.
2.2.1. Diels–Alder reactions on diene 1a
The thermodynamic, kinetic and structural results are shown in
Table 2. In agreement with the experimental data, the computa-
tional results suggest that in the oxo Diels–Alder reaction on 1a,
the most kinetically favored product is the anti-endo (Scheme 5a).
This is also the preferred adduct from a thermodynamic point of
Ts
H
CO2Et
EtO2C
O
N
R
Ts
5
O
N
O
O
H
CO2Et
CO2Et
(a)
O
O
toluene, 0.50 M
reflux
toluene, 0.50 M
reflux
R
R
O
O
7
88 %, R = Me (endo:exo 91:9)
66 %, 6 R = Me (exo:endo 7:3)
8
50 %, R = Pr
10
11 %,
R = Pr (exo:endo 7:3)
43 %, 9 R = CH2OAc
6 %, 11 R = CH2OAc (exo:endo 9:1)
R
Z
O
O
H
H
H
CO2Et
O
O
O
(b)
quant., 12 R = Br
O
toluene, 0.50 M
reflux
13
quant.,
R = CN
R = Br, CN
R
Z = O, NTs
R
Scheme 5. Substrate scope for cycloadditions using ethyl glyoxylate and N-tosylimine 5 with cis-cyclohexadienediols obtained by biotransformation using E. coli JM109
(pDTG601).
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M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
5
Ts
O
O
N
O
5
CO2Me
O
EtO
CO2Me
H
CO2Et
6.0 equiv.
H
CO2Et
6.0 equiv.
(a)
(b)
14, traces
O
toluene 0.5M
140ºC
toluene 0.25M
140ºC
O
O
O
4
83 %, 14
nOe
O
H
EtO
CO2Me
H
O
O
Scheme 6. Cycloadditions using ethyl glyoxylate and N-tosylimine 5 with cis-cyclohexadienediol 4 obtained by biotransformation using Ralstonia eutropha B9.
CO2Et
1a occur by the coupling of the dienophiles on the diene less hin-
dered face, anti to the isopropylidene group. The electron-rich neg-
atively charged zones (in red) are strongly localized over the
oxygen atoms, while electron-poor positive zones (in blue) are lar-
gely restricted to the hydrogen atoms. This charge distribution
points to significantly polar diene and dienophile molecules, whose
interaction in an apolar solvent is electrostatically driven. Indeed,
the anti-attack allows for a better fit between the negative and pos-
itive zones of the reactants, thus decreasing the electrostatic (and
steric) repulsion.
Ts
O
N
i. O3 , EtOH
HO
O
O
(a)
ii. DMS, overnight
iii. NaBH4, EtOH
O
Me
EtO2C
N
OH
OH
O
Ts Me
6
44 % overall
15
EtO2C
O
O
i. O3 , EtOH
HO
HO
ii. DMS, overnight
iii. NaBH4, EtOH
O
Me
O
It is worth analyzing the electronic features that affect the
energy of the transition structures, and with that, the stereoselec-
tivity. The perturbation theory,26 provides a method to qualita-
tively estimate the slope of an early part of the path along the
O
Me
16
7
endo
60 % overall
11.0 Hz
O
4.0 Hz
OH
H
Ts
reaction coordinate leading up to the transition state (
steeper path is likely to lead to the higher-energy transition struc-
ture. Two major factors influence the E: the Coulombic repulsion
DE). The
E
(b)
HO
O
N
H
H
H
Me
D
EtO2C
N
OH
8.0 Hz
Ts Me
or attraction between the reacting atoms and the HOMO–LUMO
interaction of the reactants. The first factor is modulated by the
atomic charges on the reacting atoms, whose values are shown
in Figure 1. The experimentally observed connectivity allows for
a complementary fit of the charges (Scheme 5a), in which the die-
nophile heteroatom is always linked to the diene quaternary car-
bon C3. The second term is controlled by the features of the
frontier orbitals involved in the cycloaddition. For both hetero
Diels–Alder reactions, the calculated HOMO/LUMO energy gap
O
O
HO
15
Scheme
7. Preparation
of
2,2,3,4,5,6-hexasubstituted
piperidine
and
tetrahydropyran.
view (see
DGreaction values), although this trend is not followed by
all the systems, thus suggesting a kinetic control of the reactions.
In fact, under this assumption, the calculated Curtin–Hammet pro-
duct ratio is 93:6 anti-endo/anti-exo, which is close to the observed
diastereomeric proportion. For the imino Diels–Alder reaction on
1a, the theoretical data points to the formation of two kinetically
favoured products, anti-exo/anti-endo, with an expected ratio of
62:38. This outcome is in agreement with the experimental
diastereomeric mixture (7:3, Scheme 4a), showing the excellent
performance of the computational model at predicting the stereos-
electivity of these hetero Diels–Alder reactions. The activation
energy for the aza Diels–Alder reaction is predicted to be higher
than the oxo one, giving an explanation for the decrease in the
yield from product 7 to 6 (Scheme 5a).
indicates
a
normal
electronic
demand
(HOMOdiene ?
LUMOdienophile, Table 3), with both molecular orbitals being cen-
tered at the reacting atoms (Fig. 2). Given the above-mentioned
connectivity, the symmetry of the frontier orbitals is in accordance
with the observed endo preference when using ethyl glyoxylate as
a dienophile, leading to an HOMO–LUMO constructive overlap.
However, the exo spatial arrangement is slightly preferred in 6
(Table 2), thus revealing that there must be some special structural
features at the transition state that explain this unexpected out-
come (see below). Overall, the obtained stereoselectivity seems
to be supported by a complex combination of electrostatic, elec-
tronic and structural factors.
It is also noteworthy that the activation free energy (D
Gà) for
the cycloadditions is significant (see Table 3). This is caused by a
combination of a negative entropic factor and the use of high tem-
peratures to allow the reactants to overcome the activation barrier,
caused mainly by a substantial structural deformation at the tran-
sition state. The interaction between this geometrical distortion
and the attractive and repulsive forces at the transition state seems
to be quite different for the various cycloadditions, since the acti-
vation free energies span a range of 35–54 kcal/mol.
2.2.2. Diels–Alder reactions on substrate 4
We performed the same computational analysis on the hetero
Diels–Alder processes using diene 4, and the results are summa-
rized in Table 2. Again, the theoretical data are in close agreement
with the experimental findings (Scheme 6a). Under kinetic control,
the reaction between 4 and ethyl glyoxylate gives rise to a unique
product anti-endo 14, with an activation energy significantly lower
than for the other possible Diels–Alder adducts. Conversely, when
dienophile 5 is used, no imino Diels–Alder product is observed. The
computational results show an increase of 10.9 kcal/mol in the
Figure 1 shows the optimized geometries and charge distribu-
tion of diene 1a and both dienophiles. As expected by the bulky
nature of the isopropylidene group, both Diels–Alder reactions on
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6
M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
Table 2
D
G values (under the experimental conditions), activation energy and products ratio for the Diels–Alder reactions
System
Product
D
Greaction (kcal/mol)
Ea (kcal/mol)
Product ratioa
Structural parameterse
D
d
Dx (eV)
XOÁ Á ÁO (Å)b
XC–HÁ Á ÁO (Å)c
Xp (Å)d
Diene 1a
Ethyl glyoxylate
anti-endo 7
anti-exo 7
syn-endo
syn-exo
anti-exo 6
anti-endo 6
syn-endo
syn-exo
6.9
8.1
8.3
13.0
14.0
8.9
17.2
19.8
16.8
18.7
20.2
25.9
19.4
19.5
30.8
33.2
93
6
1
5.40
4.80
4.55
3.70
4.67 (2)
4.62 (3)
4.01 (5)
3.74 (4)
2.74 (3)
2.55 (3)
2.85 (3)
2.48 (3)
2.55 (3)
2.61 (5)
2.47 (5)
2.44 (3)
2.22 (1)
2.88 (2)
2.63 (2)
2.74 (2)
6.04 (3)
5.63 (2)
5.46 (3)
5.14 (3)
0.19
0.18
0.08
0.18
0.85
1.02
0.88
0.11
1.21
1.38
0
Dienophile 5
62
38
0
0
Diene 4
Ethyl glyoxylate
anti-endo 14
anti-exo
syn-endo
syn-exo
anti-endo
syn-exo
anti-exo
9.6
9.9
10.8
14.2
10.4
9.7
18.6
21.7
22.2
28.2
29.5
30.8
30.9
34.9
97
2
1
5.2
4.5
5.5
4.9
3.24 (3)
3.15 (2)
3.37 (2)
3.54 (3)
2.77 (2)
2.66 (3)
2.75 (3)
2.62 (4)
2.52 (5)
2.45 (3)
2.55 (3)
2.54 (5)
2.42 (2)
2.81 (2)
2.61 (2)
2.81 (3)
6.71 (3)
5.74 (3)
6.62 (3)
6.23 (3)
0.11
0.18
0.01
0.15
1.11
0.41
0.22
0.33
1.00
1.17
0
Dienophile 5
75
15
10
0
9.5
10.7
syn-endo
The structural parameters and asynchronicity (Dd) of the transition states and the electrophilicity difference of the reactants (Dx) are also listed.
a
According to the Curtin–Hammet principle.
XOÁ Á ÁO denotes the average distance of disfavorable OÁ Á ÁO interactions between both reactants moieties. For the systems containing the dienophile 5, only the interactions
b
below 5 Å were taken into account.
c
XC–HÁ Á ÁO represents the average distance of favorable C–HdieneÁ Á ÁOdienophile interactions below 3 Å.
d
Xp estimates the average distance of disfavorable interactions between the positively charged bulky groups of both reactants moieties. For the ethyl glyoxylate-containing
systems, Xp represents the mean distance of HÁ Á ÁH interactions (<3 Å). For the rest of the systems, Xp is calculated as the average CÁ Á ÁC distance (<7 Å) between the –CH2–
ester group and the sulfur-attached carbon atom of the tosyl group in 5, and the methyl group from the ester moiety in diene 4, the aromatic methyl group of diene 1a, and
the isopropylidene quaternary carbon atom of both dienes.
e
The number of the interactions considered are shown between brackets.
Table 3
Activation free energy (under the experimental conditions) and HOMO–LUMO energy gap for the studied hetero Diels–Alder processes
System
Product
D
Gà (kcal/mol)
HOMO–LUMO energy gap (kcal/mol)
Diene ? dienophile Dienophile ? diene
Diene 1a
Ethyl glyoxylate
Dienophile 5
anti-endo 7
anti-exo 7
syn-endo
syn-exo
anti-exo 6
anti-endo 6
syn-endo
syn-exo
anti-endo 14
anti-exo
syn-endo
syn-exo
35.4
37.4
38.6
45.3
38.4
38.8
50.9
54.4
38.7
42.0
42.3
48.6
50.9
52.3
52.6
56.8
81.0
77.4
89.1
85.6
142.4
143.0
136.6
137.3
Diene 4
Ethyl glyoxylate
Dienophile 5
anti-endo
syn-exo
anti-exo
syn-endo
activation energy barrier of this process, which in turns explains
the need for drastic experimental conditions to ensure satisfactory
conversions. This leads to a high
which slows the reaction and allows the dienophile to first decom-
pose in the system.
The optimized geometry and electrostatic potential of 4 are
depicted in Figure 1. Given the size and charge distribution of
the ester and isopropylidene groups, significant steric and electro-
static interactions with the dienophiles are expected. Therefore,
the aza and oxo Diels–Alder attacks should proceed anti to the iso-
propylidene group, thus giving rise to the anti-endo adduct as the
kinetically favoured product. The repulsion forces are so consider-
able in the presence of 5, that the formation of the expected
Diels–Alder bicycle is precluded.
and negative atomic charges of diene and dienophiles (Fig. 1) leads
to the expected connectivity in each system. Remarkably, from 1a
to 4 the charges on the reacting carbon atoms vary to a great
extent, causing the observed connectivity to invert (compare
adducts 7 and 14). This can be rationalized by taking into account
the structural differences between both dienes. The presence of a
deactivating ester group in 4 removes electron density from the
D
Gà value (50.9 kcal/mol, Table 3),
p
system, thus increasing the charge on C2. Additionally, the elim-
ination of the aromatic methyl group from 1a allows one of the iso-
propylidene oxygen atoms to approach and donate electron
density to C5, negatively charging it. However, since there is no
great difference in the atomic charges for C2 and C5 in 4, we have
performed the same computational analysis for the reversed con-
nectivity; the results showed higher activation energy barriers
(not shown). As expected, HOMO and LUMO orbitals are localized
at the reacting atoms (Fig. 2) and, considering the observed
The electronic features were also analyzed (Table 3), and a nor-
mal electronic demand was found. Again, the best fit of the positive
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M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
7
exert those effects, it is worth analyzing the structural determi-
nants behind the kinetic preference of the reactions. To aid the dis-
cussion, the thermodynamic and structural parameters of all the
transition states can be found in Tables 2 and 3, while the opti-
mized geometries and charge distribution for the most stable of
them are depicted in Figure 3.
In general, the most stable transition states (TS) for the studied
hetero Diels–Alder reactions (Fig. 3) are able to better separate the
equally charged groups allowing to relieve the electrostatic and
steric repulsion. This is evident when looking at their structural
parameters (Table 2). For the systems containing diene 1a, the
average distance of disfavorable OÁ Á ÁO interactions between the
diene and dienophile moieties (XOÁ Á ÁO) are larger for the lowest-
energy transition states. Interestingly enough, the strength of the
C–HdieneÁ Á ÁOdienophile hydrogen bonds is not a relevant structural
determinant, since their average distance (XC–HÁ Á ÁO) does not seem
to correlate with the activation energy (Ea). When the dienophile 5
is present, it is also important to consider the repulsive interac-
tions between the positively charged bulky groups, whose mean
distance (Xp values) is higher for the observed TS. This is related
to the unexpected stability of the anti-exo TS; while it is not
favoured from an electronic point of view (destructive HOMO–
LUMO overlap), this arrangement greatly relieves the electrostatic
repulsion of the tosyl and isopropylidene groups, making it slightly
more stable than the expected anti-endo TS (see Fig. 3 and Table 2).
Likewise, a similar phenomenon occurs when 4 and 5 react, where
an endo/exo product mixture is also predicted (though it is not
experimentally evidenced because of the high-energy activation
barrier). Therefore, it can be concluded that the use of the highly
hindered dienophile 5 gives rise to a decrease in the endo/exo
diastereoselectivity.
Figure 1. DFT optimized geometries for the dienes 1a and 4, and dienophiles ethyl
glyoxylate and its N-tosyl imine 5. In all cases, the electrostatic potential is mapped
on an isodensity surface (isodensity value = 0.002 e, scale: -0.02 V (red) to 0.02 V
(blue)). The labels for the reacting atoms are also shown with the NPA atomic
charges between brackets. Color code: C (gray), H (white), O (red), N (blue), S
(yellow).
The same analysis for the cycloaddition on diene 4 is not
straightforward. Even though the same structural parameters arise
as relevant, the trends are not well defined. The preferred TS are
those that minimize the electrostatic repulsion (high XOÁ Á ÁO and
Xp values), relieving at the same time the steric hindrance through
an anti-facial attack.
In order to characterize the polarity of the Diels–Alder reac-
tions, the global electrophilicity difference (
Dx) and the asyn-
chronicity ( d) were determined (Table 2). All the reactions
D
involve an asynchronous bond formation, although this phe-
nomenon is much more important when dienophile 5 is used, with
D
d values that suggest a two-step mechanism.27 Firstly, a C–C
bond is formed through a nucleophilic/electrophilic two-center
interaction. After that, the formation of the C–N bond begins, thus
initiating the second stage of the reaction. Although there is not a
strict linear relationship, an increase in the reaction polarity seems
to be accompanied by an increase in its asynchronicity, and less
drastic experimental conditions are required.27 Indeed, the calcu-
lated average
D
d values correlate with the global electrophilicity
difference (
D
x) (see Table 2). For a given diene, the imino Diels–
Alder reaction has a more polar character than the oxo cycloaddi-
tion, but no decrease in the activation barrier was observed exper-
imentally (Table 2). This fact stresses the relevance of the
electrostatic and steric hindrance experienced by the TS when
the ester and tosyl bulky groups are present in the dienophile,
which counteracts the expected trend. Given a dienophile, diene
4 leads to a reduction in the reaction polarity, thus accounting
for the need of more drastic conditions to reach high yields (com-
pare Schemes 5a and 6a).
Figure 2. Spatial distribution of the HOMO for the dienes 1a and 4, and LUMO for
the dienophiles ethyl glyoxylate and its N-tosyl imine 5. The isodensity value is
0.0004 e. Color code: C (gray), H (white), O (red), N (blue), S (yellow).
connectivity, their constructive overlap supports the observed endo
arrangement.
2.2.3. Structural determinants
3. Conclusion
The electrostatic and steric interactions seem to have a key
impact in the yield and stereoselectivity. Both factors play a role
in modulating the structure and energy of the transition states
and products. To deeply understand how the different interactions
Herein we have studied the aza and oxo Diels–Alder cycloaddi-
tions of isopropylidene protected cis-cyclohexadienediols using
ethyl glyoxylate and its N-tosyl imine as dienophiles. The expected
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8
M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
Figure 3. DFT optimized geometries for the most stable transition states corresponding to the Diels–Alder reactions between ethyl glyoxylate and 1a (a), 5 and 1a (b), ethyl
glyoxylate and 4 (c), and 5 and 4 (d). The forming bonds are represented as dotted lines with their distances in Å. Asychronicity values ( d) are also shown. In all cases, the
D
electrostatic potential is mapped on an isodensity surface (isodensity value = 0.002 e, scale: -0.02 V (red) to 0.02 V (blue)). Color code: C (gray), H (white), O (red), N (blue), S
(yellow).
heterocyclic bicyclo[2.2.2]octanes were obtained and fully charac-
terized by spectroscopic methods. Excellent regio- and stereoselec-
tivities were found for both types of cycloadditions. Quantum
chemical calculations at the B3LYP/6-31+G(d,p) level of theory
were performed to rationalize the observed selectivities. Excellent
agreement of the used computational models with the experimen-
tal results was found, which allowed us to rationalize the experi-
mental reactivity trends. In addition, as an example of the
synthetic importance of the preparation of these bicyclo[2.2.2]oc-
tanes, the enantiopure preparation of a 2,2,3,4,5,6-hexasubstituted
piperidine and tetrahydropyran from toluene in only four synthetic
steps has been described.
are reported in ppm relative to the center line of the CDCl3 triplet
(77.0 ppm). Optical rotations were measured on a Zuzi 412
polarimeter using a 0.5 dm cell. [a] values are given in units of
D
deg cm2 gÀ1 and concentration values are expressed in g/100 mL.
Analytical TLC was performed on silica gel 60F-254 plates and visu-
alized with UV light (254 nm) and/or p-anisaldehyde in acidic
ethanolic solution. Flash column chromatography was performed
using silica gel (Kieselgel 60, EM reagent, 230–400 mesh) Chemi-
cals and reagents were purchased from Sigma–Aldrich and used
as received. E. coli JM109 (pDTG601) was generously donated by
Prof. David T. Gibson (1938–2014). All of his strain collections
are currently managed by Prof. Rebecca Parales (University of Cal-
ifornia, Davis). Ralstonia eutropha B9 was generously donated by
Prof. Andrew G. Myers (Harvard University-USA). Shake-flask culti-
vation was carried out using a Thermo Forma orbital shaker. A Sar-
torious-Biostat A plus bioreactor fitted with a 5 liter baffled vessel
was used as bioreactor.
4. Experimental
4.1. General
All solvents were distilled prior to use. Mass spectra (MS) were
recorded on a Shimadzu GC–MS QP 1100 EX instrument using the
electron impact mode (70 eV). High resolution mass spectra were
obtained on a Bruker Daltonics Q-TOF spectrometer (ESI mode),
at Polo Tecnólogico de Pando, Facultad de Química, Universidad
de la República. Infrared spectra (IR) were recorded either on neat
samples (KBr disks) or in solution on a Shimadzu FT-IR 8101A spec-
trophotometer. NMR spectra were obtained in CDCl3 on a Bruker
Avance DPX-400 or a Bruker Avance III 500 instruments as indi-
cated. Proton chemical shifts (d) are reported in ppm downfield
from TMS as an internal reference, and carbon chemical shifts
4.2. Biotransformation procedures
4.2.1. Media composition
Luria–Bertani (LB) medium used for cell growth contained:
Bacto-Tryptone (10 g/L), Bacto-Yeast Extract (5 g/L), and sodium
chloride (10 g/L). Agar (15 g/L) was added for solid media. When
needed the medium was supplemented with sterile ampicillin
sodium salt (0.1 g/L).
Mineral Salts Broth (MSB) used for the precultures for E. coli JM109
(pDTG601) contained: K2HPO4 (16 g/L), KH2PO4 (14 g/L), (NH4)2SO4
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M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
9
(5 g/L), Bacto-Yeast Extract (15 g/L); after sterilization the medium
was supplemented with sterile glucose (30 g/L), MgSO4Á7H2O (2 g/
L) and ampicillin sodium salt (0.1 g/L).
bioreactor was adjusted to 7.5. The culture broth was centrifuged
at 7000 rpm and 4 °C for 30 min, the supernatant was collected
and the cell pellet properly disposed. Centrifugation led to the
separation of the liquid paraffin (which contains no detectable
amounts products) from the aqueous phase. The latter was
properly lyophilized overnight to obtain a dry powder, which
was extracted several times with ethyl acetate until no more
diols were detected by TLC. The solvent was evaporated to afford
the corresponding diol, which was washed several times with
hexanes to remove the liquid paraffin traces.
Bioreactor cultures and biotransformations using Ralstonia eutro-
pha B9. Two 500 mL flasks containing 90 mL of LBII medium were
inoculated with a single colony of Ralstonia eutropha B9 and grown
over 48 h at 28 °C, 150 rpm. Both entire cultures were used to inoc-
ulate the bioreactor (Sartorius Biostat A plus), charged with an ini-
tial volume of 3 L, and set to 300 rpm, 30 °C, and air flow rate of 3
L/min. An initial volume of 17.5 mL of 1.5 M fructose solution was
added. The pH value was controlled automatically at 7.4 by the
addition of concentrated ammonium hydroxide during the whole
process. After 20 h a second pulse of 1.5 M fructose solution
(19 mL) was added along with a first pulse of 1.5 M sodium ben-
zoate solution (4.3 mL). From this time on, 8 mL of both solutions
(fructose and sodium benzoate) were added every 4 h over
3.5 days using a peristaltic pump. Stirring was adjusted when
needed, (based on the oxygen profile of the process) reaching at
the end 600 rpm. After this, the culture broth was centrifuged at
7000 rpm and 4 °C for 30 min, the supernatant was collected and
the cell pellets were properly disposed. The resulting media (which
contained 27 g/L of 2) was lyophilized to obtain a white solid,
which had a 79% content on diol 2 (determined by NMR using
sodium acetate as internal standard). This solid is stable and can
be stored at room temperature in a proper dessicator.
LB II used for cell growth and precultures of Ralstonia eutropha B9
contained: Tryptone (10 g/L), Yeast Extract (5 g/L), CaCl2Á2H2O (1 g/
L), MgCl2 (0.05 g/L). Agar (15 g/L) was added for solid media.
Bioreactor defined mineral salt medium for E. coli JM109
(pDTG601) consisted of: KH2PO4 (7.5 g/L), citric acid (2.0 g/L),
MgSO4Á7H2O (5.0 g/L), ferric ammonium citrate (0.3 g/L), 98%
H2SO4 (1.4 mL/L) and trace metal solution (1.5 mL/L). After steril-
ization, pH is regulated to 6.8 by the addition of conc. ammonium
hydroxide, followed by supplementation with sterile thyamine
hydrochloride (0.3 g/L) and ampicillin sodium salt (0.1 g/L). Trace
metal solution contained: citric acid (40 g/L), MnSO4Á2H2O (30 g/
L), NaCl (10 g/L), FeSO4Á7H2O (1 g/L), CoCl2Á6H2O (1 g/L), ZnSO4Á
7H2O (1 g/L), CuSO4Á5H2O (0.1 g/L), H3BO3 (0.1 g/L), NaMoO4Á2H2O
(0.1 g/L); pH was adjusted to 3.0 with ammonium hydroxide.
Bioreactor defined mineral salt medium for Ralstonia eutropha B9
consisted of: KOH (0.4 g/L), Nitriloacetic acid (0.2 g/L), MgSO4
(0.28 g/L), CaCl2Á2H2O (0.07 g/L), (NH4)Mo2O24Á6H2O (0.2 mg/L),
FeSO4 (2 mg/L), Hutner’s Metal Solution⁄ (1 mL/L), NH4SO4 (1 g/
L), KH2PO4 (2.72 g/L), Na2HPO4 (2.84 g/L).
⁄Hutner’s Metal Solution composition:28 EDTA (5 g/L), ZnSO4Á
7H2O 22 g/L, FeSO4Á7H2O (10 g/L), CuSO4 (3.9 g/L), CoNO3Á6H2O
(0.5 g/L), NaBO4Á10H2O (0.36 g/L), H2SO4 (1 mL/L), H2O up to 1L.
4.2.2. Plate preparation
E. coli JM109 (pDTG601) cells properly stored in cryovials, are
streaked into LB-agar plates (supplemented with ampicillin
sodium salt). The streaked plate is incubated at 37 °C for 24 h. Sin-
gle-cell colonies are chosen for the precultures preparation. Ralsto-
nia eutropha B9 cells properly stored in cryovials are streaked into
LBII-agar plates and incubated at 30° C over 48 h. Single-cell colo-
nies are chosen for the precultures preparation.
4.3. Synthetic protocols and compounds characterization
4.2.3. Biotransformations in bioreactors
4.3.1. (1S,6R)-Methyl 1,6-isopropylidenedioxycyclohexa-2,4-
dienecarboxylate 44h
Bioreactor cultures and biotransformations using E. coli JM109
(pDTG601). Growth and biotransformation in the bioreactor using
E.coli JM109 (pDTG601) were carried out using a modification of
our previously published procedure.3b Thus, 5 mL of LB medium
supplemented with ampicillin sodium salt (0.1 g/L) and glucose
(5 g/L) were inoculated with a single colony of E. coli JM109
(pDTG601), and grown overnight at 37 °C and 150 rpm. Two
500 mL shake-flasks containing 150 mL of MSB medium were
inoculated with 1 mL of the grown culture. These preculture
flasks were placed in an orbital shaker at 37 °C and 150 rpm, for
12 hrs. Both entire cultures were used to inoculate the bioreactor
(Sartorius Biostat A plus), charged with an initial volume of 2.5 L,
and set to 500 rpm, 30 °C, and air flow rate of 4L/min. The pH
value was controlled automatically to 6.8 by addition of conc.
ammonium hydroxide during the whole process. A pulse of
antifoam agents (Aldrich’s Antifoam Y: Silicone dispersion in
water 1:1) was added at the beginning of the run. At 6 h after
inoculation the dissolved oxygen value sharply increased
(indicating carbon deprivation), whereupon a glucose fed-batch
was started by adding glucose (0.7 g/mL solution) from an initial
rate of 0.08 mL/min to 0.54 mL/min in a 20 h period. When the
biomass concentration reached 15 g/L cdw, IPTG was added to
induce toluene dioxygenase expression (IPTG final concentration
in bioreactor of 10 mg/L), and the stirrer speed was set to
900 rpm. After the culture reached the stationary phase (c.a.
26 h, 50 g/L cdw aprox), glucose feeding was decreased to
0.25 mL/min and substrate addition was started. A solution of the
corresponding substrate in liquid paraffin (0.5 M) was added at a
The lyophilized culture medium was cooled to 0 °C, suspended
on dimethoxypropane and 6 equiv of TFA were added. The reaction
mixture was stirred at room temperature for 24 h after which it
was filtered through a pad of Celite. After solvent evaporation in
vacuo, a solution of diazomethane in diethylether (3 equiv) was
added dropwise. The product was purified by column chromatog-
raphy using SiO2 as the stationary phase. Diazomethane ether solu-
tion was prepared by the decomposition of N-nitroso-N-
methylurea using a bi-phasic system oh KOH 10% and diethyl
ether. 1H NMR (400 MHz, CDCl3) d 6.18–6.09 (m, 2H), 6.08–6.01
(m, 1H), 5.88–5.80 (m, 1H), 4.98 (dd, J = 4.3, 0.7 Hz, 1H), 3.81 (s,
3H), 1.46 (s, 3H), 1.44 (s, 3H); 13C NMR (100 MHz, CDCl3) d 172.2,
124.7, 124.5, 124.1, 124.0, 106.8, 79.4, 72.7, 53.0, 26.9, 25.2.
4.3.2. General procedure for the oxo Diels–Alder reaction
To a 0.5 M solution of the corresponding diene in dry toluene
under an N2 atmosphere, ethyl glyoxylate (1.5 equiv, except for
diene 4 where 8 equiv were used) was added and the reaction mix-
ture heated at reflux until consumption of the starting material.
The reaction was monitored by thin layer chromatography. After
the starting material had disappeared, the toluene was evaporated
in vacuo. The product was purified by column chromatography
using SiO2 as stationary phase.
4.3.2.1. (1S,3S,4R,7S,8S)-Ethyl 7,8-isopropylidenedioxy-1-methyl-
2-oxabicycle[2.2.2]-5-octen-3-carboxylate endo-7.
1H NMR
(500 MHz, CDCl3) d (ppm) = 6.18–6.10 (m, 2H), 4.42 (ddd, J = 7.1,
3.6, 1.0 Hz, 1H), 4.21–4.14 (m, 3H), 4.07 (dd, J = 7.1, 0.9 Hz, 1H),
3.48 (ddt, J = 7.6, 3.7, 1.9 Hz, 1H), 1.58 (s, 3H), 1.32 (s, 3H), 1.31
flow rate of 20 mL/min using
a peristaltic pump. After the
biotransformation was completed, the pH of the medium in the
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10
M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
(s, 3H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 171.3,
135.8, 128.3, 109.6, 80.6, 75.5, 73.1, 70.5, 61.1, 39.0, 25.6, 25.3,
21.3, 14.2; IR (cmÀ1) = 2980, 2936, 2899, 1759, 1726, 1205, 1173,
1063; MS (EI, 70 eV) m/z (rel. int.): 73.05 (100), 81.10 (44), 97.10
(70), 256.20 (20); HRMS (ESI+) calcd for C14H20O5 = 291.1203 (M
sulphonylisocyanate) (final p-toluenesulphonylisocyanate concen-
tration = 2 M). The mixture was heated at reflux for 4.5 h, after
which it was cooled to 50 °C and used without any purification
to the cycloaddition reaction. A 0.5 M solution of the correspond-
ing diene in dry toluene was added to the freshly prepared N-tosyl
imine. The mixture was heated at reflux until the starting material
was consumed. The system was allowed to reach room tempera-
ture. Toluene was removed in vacuo and product was purified by
chromatographic column using SiO2 as the stationary phase.
+Na+) found 291.1277; [
a
]
D
21 = À51 (c 0.9, MeOH).
4.3.2.2. (1S,3S,4R,7R,8S)-Ethyl 7,8-isopropylidenedioxy-1-methyl-
2-oxabicycle[2.2.2]-5-octen-3-carboxylate exo-7.
1H NMR
(500 MHz, CDCl3) d (ppm) = 6.34 (ddd, J = 8.0, 6.7, 0.9 Hz, 1H),
6.10 (ddd, J = 8.0, 1.1, 1.1 Hz, 1H), 4.30–4.16 (m, 4H), 3.86 (d,
J = 2.0 Hz, 1H), 3.37–3.33 (ddt, J = 7.0, 3.6, 1.7 Hz 1H), 1.56 (s,
3H), 1.32–1.23 (m, 9H); 13C NMR (125 MHz, CDCl3) d 171.3,
134.9, 131.0, 109.0, 80.2, 73.7, 72.8, 71.8, 61.3, 38.3, 25.6, 25.2,
21.2, 14.2.
4.3.3.1. (1S,4S,7R,8S)-Ethyl 7,8-isopropylidendioxy-1-methyl-2-
tosyl-2-azabicycle[2.2.2]5-octenen-3-carboxylate exo and endo-
6. exo-Isomer: 1H NMR (400 MHz, CDCl3): d(ppm) = 7.96 (dt,
J = 1.9, 8.3 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H); 6.22 ddd, J = 0.9, 6.7,
8.0 Hz, 1H), 5.96 (dt, J = 1.1, 8.1 Hz, 1H), 4.36 (ddd, J = 0.7, 3.5,
7.1 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 4.24 (d, J = 3.3 Hz, 1H), 4.16
(dd, J = 0.9, 7.1 Hz, 1H), 3.40 (dddd, J = 1.2, 3.4, 3.4, 6.7 Hz, 1H),
2.42 (s, 3H), 1.44 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.25 (s, 3H), 1,23
(s, 3H); 13C NMR (100 MHz, CDCl3): d(ppm) = 170.7, 135.7, 129.5,
128.0, 110.0, 81.0, 73.6, 61.7, 58.9, 58.7, 37.6, 25.5, 25.3, 21.6,
19.2, 14.2; endo-isomer: 1H NMR (400 MHz, CDCl3): d(ppm)
= 8.09 (dt, J = 1.9, 8.3 Hz, 2H), 7.30–7.28 (m, 2H); 6.07 (ddd,
J = 0.9, 6.4, 7.6 Hz, 1H), 5.91 (ddd, J = 1.0, 1.6, 8.0 Hz, 1H), 4.60 (d,
J = 2.4 Hz, 1H), 4.58 (dd, J = 1.0, 7.1 Hz, 1H), 4.53 (ddd, J = 1.0, 3.3,
7.1 Hz, 1H), 4.25–4.23 (dd, J = 0.9, 7.1 Hz, 1H), 3.52–4.48 (m, 1H),
2.42 (s, 3H), 1.39 (s, 3H) 1.32–1.27 (m, 9H); 13C NMR (100 MHz,
CDCl3): d(ppm) = 170.72, 143.49, 140.28, 136.43, 129.56, 129.46,
128.13, 109.64, 79.07, 76.04, 61.48, 58.78, 56.27, 39.75, 29.69,
26.90, 25.48, 22.66, 19.23; IR (/cm): 2986, 2937, 1746, 1375,
1118, 712; MS: m/z (Int. Rel) = 65.1 (22); 91.1 (98); 155.1 (58);
248.1 (100); 406.1 (6); HRMS (ESI+) calcd for C21H27NO6-
SNa = 444.1457 (M+Na+) found 444.1451.
4.3.2.3. (1S,3S,4R,7S,8S)-Ethyl 7,8-isopropylidenedioxy-1-propil-
2-oxabicycle[2.2.2]-5-octen-3-carboxylate 8. 1H NMR (400 MHz,
CDCl3) d (ppm) = 6.19 (d, J = 8.4 Hz, 1H), 6.11 (dd, J = 8.4, 6.3 Hz,
1H), 4.39 (dd, J = 7.1, 3.5, 1.0 Hz, 1H), 4.17–4.06 (m, 3H), 3.43
(ddd, J = 7.3, 3.5, 1.7 Hz, 1H), 1.33 (s, 3H), 1.95 (ddd, J = 14.2,
11.8, 4,9 Hz, 3H), 1.71 (d, J = 4.6 Hz, 1H), 1.73–1.66 (m, 1H), 1.66–
1.59 (m, 1H), 1.57–1.49 (m, 1H), 1.28 (s, 3H), 1.27 (s, 3H), 1.23 (t,
J = 7.1 Hz, 3H) 0.98 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d (ppm) = 171.4, 134.3, 128.5, 109.5, 79.7, 75.6, 75.4, 70.3, 60.9,
38.9, 36.9, 25.6, 25.2, 16.5, 14.5, 14.1; IR (cmÀ1): 2978, 2961,
2936, 2911, 2876, 1759, 1728, 1373, 1207, 1165, 1072, 1030; MS
(EI, 70 eV) m/z (%): 91.10 (44), 123.15 (100), 165.10 (84), 295
(M+, 16); [
a
]
D
21 = À7.4 (c 1.1, MeCN).
4.3.2.4. (1S,3S,4R,7S,8S)-Ethyl-7,8-isopropylidenedioxy-1-(ace-
toxymethyl)-2-oxabicyclo[2.2.2]-5-octen-3-carboxylate 9. 1H
NMR (400 MHz, CDCl3) d (ppm) = 6.16 (d, J = 1.4 Hz, 1H), 6.15
(dd, J = 1.8, 1.1 Hz, 1H), 4.52 (dd, J = 12.2, 1.5 Hz, 1H), 4.42 (ddd,
J = 7.1, 3.0, 1.0 Hz, 1H), 4.31 (dd, J = 12.2, 1.4 Hz, 1H), 4.27 (dd,
J = 7.1, 1.1 Hz, 1H), 4.17 (t, J = 1.6 Hz, 1H), 4.11 (ddd, J = 7.1, 6.5,
1.6 Hz, 1H), 3.47 (dtd, J = 6.4, 3.4, 1.7 Hz, 1H), 2.10–2.09 (m, 3H),
2.08–1.99 (m, 1H), 1.25 (t, J = 1.5 Hz, 6H), 1.20 (td, J = 7.1, 1.6 Hz,
3H); 13C NMR (100 MHz, CDCl3) d (ppm) = 170.8, 170.7, 131.4,
129.2, 110.1, 75.9, 75.0, 74.2, 70.0, 64.6, 61.1, 39.6, 25.5, 25.2,
20.8, 14.1; IR (cmÀ1): 2984, 2937, 1748, 1242, 1209, 1057; MS
(EI, 70 eV) m/z (rel. int.): 107.1 (70), 153.1 (100), 195.1 (72),
208.1 (51), 212.1 (M+, 51); HRMS (ESI+) calcd for C14H20O5-
4.3.3.2. (1S,4S,7R,8S)-Ethyl 7,8-isopropylidendioxy-1-propyl-
2-tosyl-2-azabicyclo[2.2.2]5-octene-3-carboxylate 10 exo- and
endo-10. exo-isomer: 1H NMR (400 MHz, CDCl3) d (ppm) = 7.84
(ddd, J = 8.4, 2.1, 1.8 Hz, 2H), 7.33–7.29 (m, 2H), 6.04 (ddd, J = 1.2,
7.0, 7.9 Hz, 1H), 5.82 (dt, J = 1.0, 8.0 Hz, 1H), 4.43 (dd, J = 7.0,
1.0 Hz, 1H), 4.34–4.28 (m, 3H), 4.17 (d, J = 3.5 Hz, 2H), 3.37 (dddd,
J = 6.9, 3.5, 3.5, 1.2 Hz, 1H), 2.44 (s, 3H), 2.23–2.14 (m, 1H), 2.03–
1.91 (m, 1H), 1.50–1.40 (m, 2H), 1.27 (s, 3H), 1.26 (t, J = 7.2 Hz,
3H);13C NMR (100 MHz, CDCl3) d (ppm) = 172.1, 170.6, 131.1,
129.8, 111.4, 84.4, 77.4, 77.0, 76.7, 76.1, 69.6, 68.4, 61.4, 53.0,
38.8, 25.9, 25.7, 14.2.; endo-isomer: 1H NMR (400 MHz, CDCl3) d
(ppm) = 8.02 (ddd, J = 8.4, 2.1, 1.9 Hz, 2H), 7.34–7.29 (m, 2H),
6.12 (s, 1H), 6.11 (s, 1H), 4.65 (d, J = 2.9 Hz, 1H), 4.62 (d,
J = 6.8 Hz, 1H), 4.42 (d, J = 7.0, 3.3 Hz, 1H), 4.16–4.11 (m, 2H),
3.50 (m, 1H), 2.44 (s, 3H), 2.23–2.14 (m, 1H), 2.03–1.91 (m, 1H),
1.50–1.40 (m, 2H), 1.27 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H), 1.26–1.24
(m, 3H), 0.67 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm) = 170.6, 133.9, 129.4, 129.3, 128.3, 109.4, 79.1, 75.9, 63.9,
61.7, 57.0, 39.4, 34.6, 34.2, 26.9, 25.0, 21.6, 14.3, 14.1;IR (cmÀ1):
2974, 2937, 1751, 1373, 1352, 1325, 1209, 1180, 1163, 1078,
970, 710, 590, 550; MS (EI, 70 eV) m/z (rel. int.): 91.1 (79), 155.1
(85), 108.1 (35), 236.1 (34), 276.1 (97), 294.2 (37), 318.1 (100);
HRMS (ESI+) calcd for C23H21NO6SNa = 472.1764 (M+Na+) found
472.1764.
Na = 349.1258 (M+Na+) found 349.1334;
MeOH).
[a
]
D
21 = À23.6 (c 1.2,
4.3.2.5. (1S,3S,4S,7R,8S)-3-Ethyl -8-methyl-7,8-isopropylidene-
dioxy-2-oxabicycle[2.2.2]5-octene-3,8-dicarboxylate 14. 1H NMR
(400 MHz, CDCl3) d (ppm) = 6.46 (ddd, J = 7.3, 5.6, 1.5 Hz, 1H),
6.25 (t, J = 7.3 Hz, 1H), 5.02 (d, J = 3.8 Hz, 1H), 4.89 (dd, J = 5.3,
1.3 Hz, 1H), 4.23 (d, J = 1.6 Hz, 1H), 4.18 (dq, J = 7.3, 1.3 Hz, 2H),
3.85 (s, 3H), 3.62 (dddd, J = 6.4, 4.0, 1.7, 1.7 Hz, 1H), 1.33 (s, 3H),
1.27 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
(ppm) = 172.1, 170.6, 131.1, 129.8, 111.4, 84.4, 77.4, 77.0, 76.7,
76.1, 69.6, 68.4, 61.4, 53.0, 38.8, 25.9, 25.7, 14.2.; IR (cmÀ1):
2988, 2955, 2940, 1759, 1738, 1375, 1267, 1228, 1211, 1179,
1163, 1113, 1065, 1045, 1026, 897, 872, 856, 725; MS (EI, 70 eV)
m/z (%): 60.05 (73), 73.05 (100), 83.15 (66), 97, 10 (55), 129.15
(54), 213,20 (14); HRMS (ESI+) calcd for C15H20O7 = 335,1090 (M
4.3.3.3. (1R,4S,7R,8S)-Ethyl 1-(acetoxymethyl)-7,8-iopropylidene-
dioxy-2-tosyl-2-azabicycle[2.2.2]5-octen-3-carboxylate 11. 1H
NMR (400 MHz, CDCl3): d (ppm) = 7.93 (d, J = 8.3 Hz, 2H), 7.27–
7.31 (m, 2H), 6.26 (ddd, J = 0.9, 6.7, 8.0 Hz, 1H), 5.95 (dt, J = 1.0,
8.0 Hz; 1H), 4.95 (d, J = 13.0 Hz; 1H), 4.60 (dd, J = 1.0, 7.1 Hz, 2H),
4.28–4.38 (m, 4H), 4.26 (d, J = 12.9 Hz, 1H), 3.49 (dddd, J = 1.2,
3.4, 3.4, 7.0 Hz, 1H), 2.42 (s, 3H), 1.64 (s, 3H), 1.36 (t, J = 7.1 Hz;
3H), 1.23 (s, 6H); 13C NMR (100 MHz, CDCl3): 170.3; 170.2;
+) found 335.1101; [a]
21 = +162 (c 0.5, MeOH).
D
4.3.3. General procedure for the aza Diels–Alder reaction
The ethyl glyoxylate N-tosyl imine 5 was prepared using the
procedure described by Le Goffic et al.29 Ethyl glyoxylate was dis-
solved in dry toluene; followed by p-toluenesulphonyl isocyanate
(1 equiv) and catalytic amounts of AlCl3 (0.5% m/m of p-toluene-
Please cite this article in press as: Pazos, M.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.10.015

M. Pazos et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
11
143.7; 137.8; 131.7; 130.1; 129.4; 128.4; 110.4; 77.4; 77.1; 76.8;
76.2; 73.2; 61.5; 60.8; 58.4; 37.6; 25.5; 25.4; 21.5; 20.1; 14.2; IR
(cmÀ1): 2984, 2938, 1748, 1358, 1381, 1370, 1327, 1242, 1211,
1190, 1163, 1079, 1067, 966, 914, 882, 868, 733, 714, 669, 650,
588, 552; MS (EI, 70 eV) m/z (int. rel.): 91.1 (95), 155.1 (50),
196.1 (80), 224.1 (28), 306.1 (100), 324.2 (69); HRMS (ESI+) calcd
for C23H29NO8SNa = 502.1512, (M+Na+) found 502.1506.
nate. The animation of this negative normal mode was examined
in each case, verifying that it reflects the change in geometry in
going from reactants to products.
For the molecular modeling approach to the Diels–Alder
cycloaddition reactions, different initial geometries of the reac-
tants and products were built using the graphical model builder
of Hyperchem,30 and minimized interactively by means of the
PM3 semiempirical method. Then, all the geometries were fully
optimized employing the density functional theory method,31 with
the B3LYP hybrid functional32 and the 6-31+G(d,p) basis set, as
implemented in Gaussian 09.33 This choice was supported by the
wealth of computational literature on the Diels–Alder reactions,
which points to this functional and similar basis sets to obtain con-
sistent energetics.34 The polarization function on hydrogen atoms
was added for a better description of the hydrogen-mediated inter-
actions involved in the Diels–Alder mechanism,35 thus enhancing
the reliability of the theoretical model in assessing the energetic
and structural parameters that modulate the stereoselectivity.
Starting from the optimized geometries of the products, the transi-
tion state structures were located by a series of standard first-order
saddle-point optimizations, in which the forming C–C, C–O, and C–
N bonds were initially fixed at various lengths.
4.3.4. General procedure for the reductive ozonolysis
In a round-bottomed flask under N2, the corresponding bicycle
was dissolved in dry CH2Cl2 (up to a 0.07 M concentration). After
the addition of pyridine (3 equiv), the solution was cooled to
À78 °C and O3 was bubbled until the disappearance of the starting
material. The reaction mixture was then allowed to reach room
temperature, diluted with CH2Cl2 and a saturated solution of
NaHCO3 was added. The aqueous phase was extracted three times
with CH2Cl2. The combined organic phases were dried over Na2SO4
and the solvent was removed under reduced pressure.
4.3.5. General procedure for the reduction with NaBH4
To a 0.1 M solution of the starting material in absolute ethanol
at 0 °C, 4 equiv of NaBH4 were added. The reaction mixture was
stirred at room temperature overnight, after which an NH4Cl satu-
rated solution was added. After stirring for 10 min, the product was
extracted 4 times with a 1:1 mixture of AcOEt/isopropanol. Purifi-
cation was carried out by column chromatography using SiO2 as
the stationary phase.
The natural population analysis (NPA) phase of the natural bond
orbital analysis (NBO) was used to estimate the atomic charges.36
Zero-point energies and thermal corrections were taken from
unscaled analytical vibrational frequencies. The products ratio
under kinetic control was calculated according to the Curtin–Ham-
met principle.37 The polarity of the Diels–Alder reactions were
4.3.5.1. (2R,3R,4S,5R,6S)-Ethyl 4,5-dihydroxy-3,6-bis(hydrox-
ymethyl)-6-methyl-1-tosylpiperidine-2-carboxylate 15. 1H NMR
(400 MHz, CDCl3) d 7.81 (d, J = 8.3 Hz, 1H), 7.31–7.27 (m, 1H),
4.56 (dd, J = 8.2, 3.9 Hz, 1H), 4.50 (d, J = 11.1 Hz, 1H), 4.31–4.22
(m, 3H), 4.05 (d, J = 8.2 Hz, 1H), 3.98 (dt, J = 11.7, 3.8 Hz, 1H),
3.88 (ddd, J = 11.7, 8.8, 5.9 Hz, 1H), 3.73 (dd, J = 12.4, 9.3 Hz, 1H),
3.10 (dd, J = 9.4, 5.5 Hz, 0H), 2.53–2.43 (m, 1H), 2.41 (s, 1H), 1.60
(s, 1H), 1.34 (t, J = 7.1 Hz, 1H), 1.22 (s, 1H), 0.92 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 173.2, 143.5, 139.4, 129.6, 127.6, 108.2, 78.8,
71.7, 66.7, 63.6, 62.2, 61.7, 56.7, 38.3, 24.8, 23.8, 23.3, 21.5, 14.0;
IR (cm-1): 3476, 2884, 2940, 1738, 1329, 1260, 1159, 1088, 1040,
995, 880, 816, 548; MS (EI, 70 eV) m/z (int. rel.): 91.1 (100),
124.1 (73), 154.1 (56), 155.1 (53), 278.1 (93), 368.1 (28), 384.2
(57), 426.2 (75), 442.2 (15); HRMS (ESI+) calcd for C21H31NO8
assessed through asynchronicity (
Dd) and global electrophilicity
(x
) parameters.38
Acknowledgments
The authors would like to thank the Agencia Nacional de Inves-
tigación e Innovacion (Project FCE 6045) for financial support and
for a Ph.D. scholarship for Mariana Pazos. We would also like to
thank the Comisión Sectorial de Investigación Científica—Universi-
dad de la República for a MSc scholarship for María Agustina Vila.
We thank Prof. Alejandra Rodríguez (Facultad de Química, Polo
Tecnológico de Pando - UdelaR) for the High Resolution Mass Spec-
troscopy analysis and Prof. Guillermo Moyna and Gualberto Bottini
(Departamento de Química del Litoral, CENUR Litoral Norte—Ude-
laR) for the NOE experiments and helpful discussions regarding the
stereochemistry of the Diels–Alder cycloadducts. We also would
like to thank Lucía Almeida and Gonzalo Macias for the NMR spec-
tra of product exo-7.
SNa = 480.1668, (M+Na+) found 480.1663; [
a]
21 = +17.1 (c 0.76,
D
MeCN).
4.3.5.2. (2S,3S,4S,5R,6S)-2,5,6-Tris(hydroxymethyl)-3,4-isopopili-
denedioxy-2-methyltetrahydro-2H-pyran-3,4-diol 16. 1H NMR
(400 MHz, CDCl3)
d
4.59 (dd, J = 6.9, 5.1 Hz, 1H), 4.04 (d,
References
J = 6.9 Hz, 1H), 3.91 (dt, J = 8.4, 4.2 Hz, 1H), 3.86 (d, J = 6.4 Hz,
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1H), 1.55 (s, 3H), 1.36 (s, 3H), 1.34 (s, 3H); 13C NMR (100 MHz,
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Please cite this article in press as: Pazos, M.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.10.015