Absolute configuration of natural diastereoisomers of 6β- hydroxyhyoscyamine by vibrational circular dichroism

Article abstract of DOI:10.1021/np060133j

The absolute configuration of the two natural diastereoisomers of 6β-hydroxyhyoscyamine has been determined using vibrational circular dichroism (VCD) spectroscopy. The predicted VCD and IR spectra of (3R,6R,2′S)-6β-hydroxyhyoscyamine (1) and (3S,6S,2′S)-6β- hydroxyhyoscyamine (2) were calculated using density functional theory (DFT) with the B3LYP functional and 6-31G(d) basis set and considering the eight lower energy conformations of each diastereoisomer. In both cases, the first four conformers showed the N-Me group in the syn orientation, permitting the formation of a hydrogen bond between the hydroxy group at the tropane ring and the tertiary nitrogen atom. In addition the eight conformers showed an intramolecular hydrogen bond between the hydroxy and carbonyl groups of the tropic ester moiety. The calculated IR spectra of both molecules showed good agreement with the experimental spectra, while comparison of the experimental and calculated VCD spectra showed that the absolute configuration of dextrorotatory 6β-hydroxyhyoscyamine is (3R,6R,2′S), while the levorotatory isomer is (3S,6S,2′S).

Full text of DOI:10.1021/np060133j

J. Nat. Prod. 2006, 69, 1335-1340
1335
Absolute Configuration of Natural Diastereoisomers of 6â-Hydroxyhyoscyamine by Vibrational
Circular Dichroism
Marcelo A. Mun˜oz,† Orlando Mun˜oz,† and Pedro Joseph-Nathan*,‡
Departamento de Qu´ımica, Facultad de Ciencias, UniVersidad de Chile, Casilla 653, Santiago, Chile, and Departamento de Qu´ımica,
Centro de InVestigacio´n y de Estudios AVanzados del Instituto Polite´cnico Nacional, Apartado 14-740, Me´xico, D. F., 07000 Me´xico
ReceiVed March 23, 2006
The absolute configuration of the two natural diastereoisomers of 6â-hydroxyhyoscyamine has been determined using
vibrational circular dichroism (VCD) spectroscopy. The predicted VCD and IR spectra of (3R,6R,2′S)-6â-hydroxy-
hyoscyamine (1) and (3S,6S,2′S)-6â-hydroxyhyoscyamine (2) were calculated using density functional theory (DFT)
with the B3LYP functional and 6-31G(d) basis set and considering the eight lower energy conformations of each
diastereoisomer. In both cases, the first four conformers showed the N-Me group in the syn orientation, permitting the
formation of a hydrogen bond between the hydroxy group at the tropane ring and the tertiary nitrogen atom. In addition
the eight conformers showed an intramolecular hydrogen bond between the hydroxy and carbonyl groups of the tropic
ester moiety. The calculated IR spectra of both molecules showed good agreement with the experimental spectra, while
comparison of the experimental and calculated VCD spectra showed that the absolute configuration of dextrorotatory
6â-hydroxyhyoscyamine is (3R,6R,2′S), while the levorotatory isomer is (3S,6S,2′S).
Anisodamine, or 6â-hydroxyhyoscyamine, is a well-known
tropane alkaloid found in the roots and leaves of various species
of Physochlaina, Scopolia, Duboisia, and Datura genera.1 It
possesses a number of pharmacological uses, e.g., against cardio-
vascular disorders,2,3 acute ischemic renal failure,4 and snakebites.5
Some interest has been shown for the separation and analysis of
this drug in different matrixes such as vegetable material,6,7
pharmaceutical samples,8,9 and urine10 using different chromato-
graphic methodologies. While pharmaceutical formulations com-
prise the four possible stereoisomers,11 only (3R,6R,2′S)-6â-
hydroxy-hyoscyamine (1) and (3S,6S,2S)-6â-hydroxy-hyoscyamine
(2), also known as 7â-hydroxyhyoscyamine12 and 6â-hydroxy-
hyoscyamine,13 respectively, have been isolated from plant mate-
rial.12 These two molecules have the same (S) absolute configuration
at the tropic ester moiety,14 like other related alkaloids, and therefore
they differ only in the relative position of the hydroxy group
attached to the tropane ring. The C-3 stereocenter is responsible
for the diastereoisomeric relationship of these two molecules.
and the other dextrorotatory ([R]D ) +1.2), when measured as free
bases.12 Although the hydrolyses of these alkaloids were conducted,
and the absolute configuration of (+)-6â-hydroxyhyoscyamine and
(-)-6â-hydroxyhyoscyamine were defined as (3R,6R) and (3S,6S),
respectively, some confusion has arisen from the specific rotation
values of the two obtained tropanediols. The reported values of
[R]D ) -1.8 and [R]D ) -1.0 for the hydrochlorides of the diols
obtained from levorotatory and dextrorotatory 6â-hydroxyhyos-
cyamine, respectively, do not provide sufficient information to
establish the absolute configuration of these tropane alkaloids.
Owing to the confusing information, we decided to use an
independent contemporary methodology to establish the absolute
configuration of these molecules. This methodology can further be
useful for the absolute configuration determination of related tropane
alkaloids, like those isolated from the bark of Erythroxylum
Vaccinifolium, for which no absolute configuration is discussed.25
Recently, vibrational circular dichroism (VCD) spectroscopy has
been shown to be an excellent and reliable technique to ascertain
the absolute configuration of chiral molecules.26 The methodology
involves the calculation of all significantly populated conformations
followed by generation of the weighted vibrational spectra using
ab initio calculations,27 which are in turn compared to the
experimental VCD spectra.28 The computational time needed for
these calculations has limited the use of VCD data of natural
products, due to the relative size and complexity of these molecules,
with the exception of a few recently reported examples.29-32 Also,
most of the applications of VCD spectroscopy on organic molecules
dealt with the differentiation of enantiomeric species.33-38 The use
of these calculations for the differentiation of diastereoisomeric pairs
is very restricted38-40 and has not yet been applied in the natural
products field. Here, we present the first use of experimental and
The absolute configuration of tropane alkaloids of this type is
commonly established using chemical correlation with the corre-
sponding 3R,6â-tropanediol of known configuration.15,16 The
absolute configuration of 3R,6â-tropanediol was established17,18 as
(3R,6R) for the levorotatory free base ([R]D ) -23.3) and (3S,6S)
for the enantiomer ([R]D ) +24.1) adopting the lactone rule of
Hudson.19 Also, a levorotatory form of 6â-hydroxyhyoscyamine
hydrobromide ([R]D ) -10.5) was correlated with (+)-3R,6â-
tropanediol hydrobromide ([R]D ) +1.4) and assigned the (3R,6R)
absolute configuration.20 The same authors corrected the absolute
configurations of both enantiomeric 3R,6â-tropanediols by using
chemical correlation with (S)-methoxysuccinic acid, leading to the
opposite conclusion.21,22 Different articles concerning the isolation
of (-)-6â-hydroxyhyoscyamine are found, most of which do not
report specific rotation data. For a few exceptions negative values
were reported for the hydrobromide form of the alkaloid when
measured in water.23,24
In addition, diastereoisomers 1 and 2 where found in the hairy
roots of Hyoscyamus albus, one being levorotatory ([R]D ) -13.0)
* To whom correspondence should be addressed. Tel: +5255 5747 7112.
Fax: +5255 5747 7137. E-mail: pjoseph@nathan.cinvestav.mx.
† Universidad de Chile.
‡ Centro de Investigacio´n y de Estudios Avanzados del Instituto
Polite´cnico Nacional.
10.1021/np060133j CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/02/2006

1336 Journal of Natural Products, 2006, Vol. 69, No. 9
Mun˜oz et al.
Figure 1. DFT-optimized structures of the eight more stable conformers of (3R,6R)-6â-hydroxyhyoscyamine (1). For relative energies and
abundances, see Table 1.
Table 1. Calculated Relative Energies (kcal/mol) and
Abundances (%) of the Eight More Stable Conformers of 1 and
2, Using MMFF94 Systematic Search and B3LYP/6-31G(d)
Single-Point and Geometry Optimization Level of Theorya
hydrogen bond with the tertiary amine nitrogen in a five-atom
arrangement strongly depends on the N-Me orientation. While a
syn disposition of the N-Me group leaves the nitrogen electron pair
oriented to the hydroxy group, permitting the formation of the
hydrogen bond, an anti orientation precludes this association
completely. In both isomers (1 and 2) the four more stable
conformers show a syn N-Me group orientation, and therefore the
two possible intramolecular hydrogen bonds are present, while the
next four conformers show the anti N-Me group orientation with
only the tropic ester hydrogen bond. The preference for the syn
N-Me group orientation of 1 and 2 has been suggested previously
based on the 13C NMR chemical shift of the methyl group attached
to the nitrogen.41 The five-membered intramolecular hydrogen bond
to the nitrogen atom seems to contribute less than 1.5 kcal/mol to
the molecular stabilization. This is probably due to the geometry
of the atom arrangement in combination with the basis set used.
Both isomers show exactly the same conformational order in the
energy scale, confirming the relationship between the relative
position of the hydroxy group in the tropane ring and the
conformational preferences of the molecules. These eight more
significant conformations, accounting for 96.8% and 96.5% of the
total population in the first 5 kcal/mol range of 1 and 2, respectively,
were energy optimized by means of B3LYP/6-31G(d) calculations,
and the pertinent results are given in Table 1 as EOE values, together
with the corresponding values of the relative abundances of these
optimized geometries (%OE).
b
b
c
b
d
conf
EMMFF
ESP
%
SP
EOE
%
OE
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
2.02
1.14
2.29
1.41
1.54
1.90
0.84
1.46
1.97
1.39
2.81
2.00
1.47
0.87
2.30
1.53
0.00
0.20
0.01
0.51
0.92
0.74
1.66
1.30
0.00
0.46
0.23
0.71
1.25
1.94
1.23
1.82
25.54
18.28
25.02
10.85
5.39
7.30
1.54
2.85
34.88
16.13
23.54
10.43
4.20
1.32
4.40
1.61
0.00
0.08
0.24
0.25
0.87
0.93
1.12
1.30
0.00
0.23
0.25
0.43
1.19
1.24
1.27
1.42
25.66
22.42
17.11
16.83
5.91
5.34
3.88
2.85
30.45
20.66
19.97
14.74
4.09
3.76
3.57
2.76
2g
2h
a The conformers of each diastereoisomer are arranged according to
the optimized energy calculations. b Relative to the lowest energy
conformer of each diastereoisomer. c Calculated using the single-point
energies in the first 5 kcal/mol. d Calculated using the optimization
energies of the relevant conformers.
calculated VCD spectra for the determination of the absolute
configuration of two diastereoisomeric secondary metabolites.
The difficulty in distinguishing these two diastereoisomeric
molecules is due to the fact that not all bands in the VCD spectra
of the two stereoisomers are mirror images, as would be the case
for a pair of enantiomers. This implies the need to identify the
vibrational bands that are related to the asymmetric centers that
differ in the two diastereoisomers. Also, it is necessary to perform
the conformational analysis and calculation of vibrational spectra
for both stereoisomers, since this information cannot be inferred
from one molecule to the other, as in enantiomers.
Results and Discussion
The conformational distribution of 1 and 2 (Table 1), calculated
using MMFF94 systematic conformational searches followed by
single-point energy calculations at the B3LYP/6-31G(d) level of
theory, shows a clear tendency of the two hydroxy groups to form
intramolecular hydrogen bonds. The eight most populated confor-
mations of 1 and 2 shown in Figures 1 and 2, respectively, display
this type of association between the hydroxy and the carbonyl
groups on the tropic ester, forming a six-membered ring. On the
other hand, the possibility of the C-6 hydroxy group of forming a
The calculated vibrational spectra (IR and VCD) of the lowest
energy conformers of 1 and 2 using the B3LYP/6-31G(d) level of
theory are shown in Figures 3 and 4, respectively. The combination

Natural Diastereoisomers of 6â-Hydroxyhyoscyamine
Journal of Natural Products, 2006, Vol. 69, No. 9 1337
Figure 2. DFT-optimized structures of the eight more stable conformers of (3S,6S)-6â-hydroxyhyoscyamine (2). For relative energies and
abundances, see Table 1.
Figure 4. Calculated IR absortion (bottom) and VCD spectra (top)
for the eight lowest energy conformers of (3S,6S)-6â-hydroxy-
hyoscyamine (2) at the B3LYP/6-31G(d) level of theory. Half of
the spectra is offset for clarity. Frequencies are scaled by a factor
of 0.97.26
Figure 3. Calculated IR absortion (bottom) and VCD spectra (top)
for the eight lowest energy conformers of (3R,6R)-6â-hydroxy-
hyoscyamine (1) at the B3LYP/6-31G(d) level of theory. Half of
the spectra is offset for clarity. Frequencies are scaled by a factor
of 0.97.26
of DFT functionals and basis set used was selected considering its
good agreement with experiment and reasonable consumption of
computer time that this level of theory has shown in the past.42
Recently published examples30,43 confirm that indeed calculations
at the B3LYP/6-31G(d) level of theory provide a nice balance
between computing time and spectral similarity. In any event, the
use of a larger basis set will not modify the goal of this work,
which is the diastereoisomeric distinction of 1 and 2. Some
additional considerations regarding limitations imposed by very
large computing time requirements for bigger basis sets are given
in the Experimental Section, and in the Supporting Information are
shown some results obtained using the 6-311G++(d) basis set.
The calculated IR spectra do not show significant differences
between the considered conformers. In severe contrast, the VCD

1338 Journal of Natural Products, 2006, Vol. 69, No. 9
Mun˜oz et al.
Figure 5. Relative amplitudes of selected vibrational modes (values in cm-1 under each structure) for the lowest energy conformer of
1 (top) and 2 (bottom). The vibrational modes are characterized: 1040 and 1041 cm-1, asymmetric stretching between C1-N-C5,
N-C1-C2, N-C5-C4, and O-C6-C7; 1084 cm-1, asymmetric stretching between O-C6-C7 and N-C1-C2; 1124 and 1125 cm-1
,
asymmetric stretching between C2-C3-C4, C1-C2-C3, and C3-C4-C5 and symmetric stretching between O-C6-C5. Note the small
arrows pointing to the atoms of interest for a particular transition, drawn using the GaussianView software package.
spectra of the lowest energy conformers of 1 and 2 show a series
of bands that depend on the conformational preferences, particularly
of the tropic ester. Since these vibrations are related to a more
flexible part of the molecule, the correlation between theoretical
and experimental bands is expected to be less accurate than those
arising from conformationally more restricted parts of the molecule,
especially when a small-size basis set is used to calculate geom-
etries, energies, and vibrational behavior. Additionally, these bands
are not usable for absolute configuration determination of the whole
molecule since they arise from an asymmetric center with the same
configuration in both stereoisomers. Nevertheless, the spectral range
between 950 and 1100 cm-1 shows at least three bands that can be
assigned mainly to vibrations on the tropane ring (Figure 5), which
remain unchanged in all low-energy conformers. Each band is
composed by coupled interactions between different vibrations of
the bonds in the tropane ring. The most intense of these three bands
(1040 and 1041 cm-1 for 1 and 2, respectively) is due to a coupled
interaction between the four asymmetric stretching vibrations of
C1-N-C5, N-C1-C2, N-C5-C4, and O-C6-C7. The band
at 1084 cm-1 for both molecules is produced by the asymmetric
stretching of O-C6-C7 and N-C1-C2, while the band at 1125
cm-1 is due to the asymmetric vibrations of C2-C3-C4,
C1-C2-C3, and C3-C4-C5 and the symmetric stretching of
O-C6-C6. These motions are mirror images between the dia-
steroisomers, and consequently, the produced bands show opposite
intensities when comparing the spectra of 1 and 2, and therefore
can be used to assign the diastereoisomeric compounds. Since the
C-H absorption of CHCl3 occurs around 1200 cm-1, the VCD
spectra were acquired from CDCl3 solutions whereby the C-D
Figure 6. Comparison of observed and calculated IR absortion
(bottom) and VCD spectra (top) of (3R,6R)-6â-hydroxyhyos-
cyamine (1).
absorption is shifted to around 900 cm-1
.
When the calculated VCD spectra obtained from the low-energy
conformers of 1 and 2, using the Boltzmann distribution, derived
from the energies of the optimized structures, are combined for
each molecule in a single weighed plot, which is then compared
with the experimental VCD spectra, as shown in Figures 6 and 7,
respectively, the absolute configuration of both diastereoisomers
follows unequivocally. The bands assigned to the tropane ring

Natural Diastereoisomers of 6â-Hydroxyhyoscyamine
Journal of Natural Products, 2006, Vol. 69, No. 9 1339
using the Gaussian 03W software package. The conformational search
was done only with the indicated force field method, while the ab initio
calculations were performed with the described limited basis set size
and a finite integration grid without considering solvent effects. Typical
calculations required between 30 and 40 h of computational time per
conformer when using a desktop personal computer (PC) with 2 Gb
RAM operated at 3 GHz. Calculated dipole and rotational strengths
were converted to molecular absorptivities (M-1 cm-1) and then plotted
as Lorentzian bands with half-widths of 6 cm-1. The four lower energy
conformers of 1, which accounted for 82% of the conformational
population in the initial 2 kcal/mol, were also calculated at the higher
B3LYP/6-311G++(d) level of theory. On average, each conformer
required some 200 h computing time, making this an inpractical
procedure. The weighted calculated and experimental VCD curves are
compared in Figure 1 of the Supporting Information and provide further
evidence that calculations at the B3LYP/6-31G(d) level of theory are
reliable for the diastereoisomeric distinction of 1 and 2.
Preparation of 1 and 2. The two diasteroisomers of 6â-hydroxy-
hyoscyamine were prepared by catalytic hydrogenolysis of commercial
(-)-scopolamine hydrobromide (Sigma-Aldrich) with Nickel Raney
W1.44 The separation of the resulting mixture [1 (33.9%), 2 (41.7%),
and (-)-hyoscyamine (19.7%)] was made using ion pair column
chromatography with 1-heptanesulfonic acid sodium salt at pH 4 (buffer
HOAc/AcONa 50 mM)/MeOH (17:3) as mobile phase and Varian C18
(40 µm) as stationary phase in a one-step procedure. Both methodolo-
gies were optimizations of previously reported procedures.12 The three
obtained compounds showed 1H and 13C NMR chemical shifts and
specific rotations identical to those of natural samples.12
Supporting Information Available: Comparison of experimental
and calculated VCD spectra of 1 using the B3LYP/6-311G++(d) level
of theory, and DFT B3LYP/6-31G(d) atom coordinates for the eight
low-energy conformers of 1 and 2. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 7. Comparison of observed and calculated IR absortion
(bottom) and VCD spectra (top) of (3S,6S)-6â-hydroxyhyoscyamine
(2).
vibrations clearly differentiate 1 from 2, showing that the absolute
configuration of (+)-6â-hydroxyhyoscyamine (1) is (3R,6R,2′S),
and that for the diastereoisomeric (-)-6â-hydroxyhyoscyamine (2)
is (3S,6S,2′S), as previously reported.12 It further follows that, since
some early publications17,18,20 are already amended,21,22 verification
of configurational assignments, either by chemical correlation or
by VDC measurements, remains to be done in some cases.15,16,21-24
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