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3.2. Nuclear magnetic resonance analysis
29Si spectrum of compound 4a showed the presence of three
trialkylsilyl substituents (11.16, 12.42, and 13.11 ppm). The
silicon-proton HMBC spectrum of the tri-triisopropylsilyl
derivative 4a shows the clear correlation between the 29Si
resonance at 11.16 ppm and the 3Ј proton. Correlations were
not observed for the other 29Si resonance, presumably be-
cause of broadening by the conformational equilibrium in-
volving ring A causing a decay of signal intensity during the
long delays in the HMBC experiment. However, in
DMSO-d6 the slower exchange rate of the OH protons
allows their direct observation and such protons were ob-
served at 4.69 ppm, d, J ϭ 5.5 Hz (H-4Ј), 4.31 ppm, d, J ϭ
4.5 Hz (H-11), and 4.05 ppm, d, J ϭ 4.0 Hz (H-2Ј). All the
required cross-peaks to the corresponding C-H protons were
observed in the COSY spectrum. In addition, a singlet
hydroxyl was observed at 3.79 ppm consistent with the
tertiary 5- or 14-OH whereas the second tertiary hydroxyl
seems to be among four overlapping signals at 4.05. These
results require the three tri-isopropylsilyl groups to be lo-
cated at C-1, C-3Ј, and C-19.
To obtain good quality 13C NMR spectra for ouabain and
its derivatives is complicated by peak broadening and peak
suppression due to conformational equilibria [16] and dif-
ferent solubilities. Therefore, it was necessary to record
spectra at higher temperatures in DMSO-d6. Furthermore,
because of the bulky trialkylsilyl groups, steric crowding
together with overlapping signals in the proton spectra,
made assignment by correlation inaccurate. Therefore, NOE
and difference double resonance measurements from 29Si
coupling techniques were also employed to establish the
number and location of the trialkylsilyl substitution.
1
Integration of the trimethylsilyl protons in the H NMR
spectrum of the trimethylsilyl derivative 2 showed the presence
of six trimethylsilyl groups indicative of silylation of the six
primary and secondary, but not the more sterically hindered
tertiary hydroxy groups. Therefore, the 1,2Ј,3Ј,4Ј,11␣,19-
hexa-trimethylsilyl structure was assigned to this derivative.
1
Analysis of the H and 13C NMR spectra of the triiso-
propylsilyl ouabain derivative 3a showed that determination
of the number of silyl groups was not possible by proton
integration or by NOE experiments because of overlap of
the isopropylsilyl protons with signals from the steroid ring
protons. Similar problems applied to the corresponding 11-
Integration of the SiMe2 and CMe3 protons in the tert-
butyldimethylsilyl derivative 4b showed incorporation of
three silyl groups. A 29Si spectrum confirmed the presence
of three tert-butyldimethylsilyl groups (19.52, 20.42, and
20.91 ppm). The location of tert-butyldimethylsilyl substi-
tution in 4b was determined from 29Si-H coupling. NOE
difference spectroscopy from the silyl methyls, and differ-
ence double resonance from the 29Si signals confirmed the
location of the silyl derivatization at the 1, 3Ј, and 19
hydroxyl positions in compound 4b.
The spectrum of the 1,11-dioxo silyl derivative 5 showed
two new carbonyl signals in the 13C NMR spectrum one of
which was assigned to the 11-ketone by comparison with
the spectrum of 6a and 6b (see below) while the remaining
carbonyl signal was assigned to C-1. Loss of the 1␣-H and
shifts in C-2, 3, 5, and 10 in the spectrum of 1,11-diketone
5 confirm the formation of the 1-ketone. This result is
consistent with the 1-OH being silylated in the tri-triiso-
propylsilyl ouabain 4a.
oxo derivative 6a. Compound 3a showed signals in the 13
C
and 29Si spectrum indicative of a single triisopropylsilyl
group. The location of the triisopropylsilyl group is indi-
cated by a positive shift in C-19 (1.83 ppm) compared with
ouabain [16] and shifts in the H2-19 protons. The 29Si signal
(16.03 ppm in CDCl3/CD3OD) is broadened to a line width
of about 30 Hz because of ring A conformational equilibria
[16]. The location of the triisopropylsilyl group at C-19 in
compound 3a was confirmed by 29Si-H coupling and double
difference resonance measurements that showed enhance-
ment of one of the 19-H2 protons. Enhancement of only one
19-H2 reflects the dependence of the interaction upon dihe-
dral angle.
The presence of two triisopropylsilyl substituents in
compound 3b was established from the 29Si spectrum
(11.67 and 13.37 ppm in DMSO-d6) with no other modifi-
cations to the steroid structure. Difference double resonance
measurements from the 29Si resonances revealed derivati-
zation of the 3Ј-hydroxy group. Irradiation of the remaining
29Si signal in 3b, however, did not result in any changes in
The 1H and 13C NMR spectra of compound 6a are
consistent with its formation from compound 4a and are in
agreement with the analysis of the corresponding tri-
tBuMe2Si derivative 6b (see below). Thus there is loss of
H-11 in the proton spectrum and appropriate positive shifts
in C-9 and 12.
1
the H spectrum, possibly due to a low three-bond 29Si to
A 29Si spectrum of the 11-oxo derivative 6b confirms the
presence of three tert-butyldimethylsilyl groups. NOE dif-
ference spectroscopy from the silyl methyls and difference
double resonance measurements from the 29Si signals lo-
cated the silyl derivatization at the 1,3Ј, and 19-hydroxy
groups in agreement with the previous assignments of the
tri-tBuMe2Si derivative 4b and consistent with the assign-
ments for tri-triisopropylsilyl ouabain 4a.
1-H coupling constant or broadening due to conformational
processes. The presence and location of both triisopropyl-
silyl substituents in compound 3b was confirmed by a pos-
itive chemical shift in C-3Ј (1.33 ppm) and C-19 (1.70 ppm)
together with shifts in H-3Ј and H2-19 [16].
1
Analysis of the H and 13C NMR spectra of the triiso-
propylsilyl ouabain derivative 4a showed that C-1, 3Ј, and
19 were positively shifted (3.85, 1.60, and 4.24 ppm, re-
spectively) as were H-1, H-3Ј, and H2-19 indicating that the
location of a third triisopropylsilyl group was at C-1. The
Inspection of the 13C spectrum of the 11-oxo derivative
6b reveals the presence of an additional carbonyl group
relative to tri-tBuMe2Si ouabain. The absence of identifiable