MAGNETIC RESONANCE IN CHEMISTRY, VOL.

31, 94-99 (1993)

13CNMR Assignments of Brassinosteroids by

Two-Dimensional Techniques

Tetsu Ando*
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu,
Tokyo 183, Japan
Masakazu Aburatani
Research Division, Fuji Chemical Industries Ltci, 530, Chokeiji, Takaoka, Toyama 933, Japan

Nozomu Koseki, Seiichi Asakawa, Takehito Mouri and Hiroshi Abe

Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu,
Tokyo 183, Japan

IH NMR spectra of eight naturally occurring brassinosteroids, plant hormones with a steroid skeleton, and five
synthetic analogues were analysed by COSY and long-range COSY measurements. Based on the 'H resonances,
the I3C signals were assigned by a combination of C-H COSY and long-range C-H COSY spectra detecting I3C
nuclei, or by HMQC and HMBC spectra detecting 'H nuclei. Although the steroids showed complex 'H signals
around 1-2 ppm, the resonances for the two angular methyl groups and the other methyl groups in the side-chain
could be assigned unambiguously and used as a reference point for the other assignments. Strong correlation peaks
between the methyl protons and carbons connected with the protons through two or three bonds were observed in
the long-range C-H COSY or the HMBC spectra. These spectra provide valuable information for assigning the
13CNMR resonances of this class of compounds.

KEY WORDS Brassinosteroids I3C NMR 2D NMR Brassinolide Steroids

detailed 13C assignments to provide a basis for future

~ ~~

INTRODUCTION
After the identification of brassinolide (BR) in 1979,'
more than 20 brassinosteroids have been discovered as
plant hormones from pollen, immature seeds, leaves,
other plant organs, algae and cultured plant celk2 Syn-
thetic standards are important for the identification of
new brassinosteroids which occur at low concentrations
in plants, and for further biological studies. 3c'NMR is
one of the most useful analytical tools to confirm the
structures of the synthetic compounds, but relatively
few chemical shift data are available.
In this paper we report the 13C signal assignments,
using two-dimensional (2D) NMR techniques, for eight
brassinosteroids and five synthetic derivatives. These
compounds include the two historically and physiologi-
cally important brassinosteroids, BR and castasterone
(CA). In structural terms CA is a 2,3-dihydroxy-6-keto
steroid and BR is one of its lactone (namely homo-oxa)
derivatives. Typhasterol (TY) and teasterone (TE), both
3-hydroxy-6-keto steroids, are another group of brass-
inosteroids classified according to their A, B ring struc-
ture. The other naturally occurring brassinosteroids
examined comprise four analogues of CA which vary
only in their respective side-chains. The five synthetic
compounds studied are various lactone derivatives. For
each of the brassinosteroids we have carried out
* Author to whom correspondence should be addressed
structural assignments of new compounds of this
general class using NMR techniques.
EXPERIMENTAL
Chemicals
The structures of the synthetic steroids used and the
numbering system are shown in Fig. 1. The following
naturally occurring brassinosteroids were synthesized :
BR,3 CA,3 TY,3 TE,3 24-epicastasterone (EC)? homo-
castasterone (HC)," dolichosterone (DS)5 and homo-
dolichosterone (HD).5 The 6-oxa isomer (1) of BR was
produced as a by-product of BR synthesis, and purified
from the mother liquors of BR recrystallization by pre-
parative TLC. The B-homo-7-oxa derivative (2) of TY
and its 6-oxa isomer (3) were synthesized from TY. TY
was acetylated with acetic anhydride in pyridine, then
treated with trifluoroperacetic acid to convert it into
two B-homo-oxa derivatives (Baeyer-Villiger reaction).
After separation using a silica gel column, 2 and 3 were
obtained by deacetylation with sodium hydroxide in 42
and 24% yields from TY, respectively. The B-homo-7-
oxa derivative (4) of TE and its 6-oxa isomer (5) were
prepared in a similar manner starting from TE. For the
NMR analyses 50 mg of each compound were used,
except for 4 and 5 when 5 mg were used.

0749-1581/93/010094-06 $08.00 Received 22 October 1991

0 1993 by John Wiley & Sons, Ltd. Accepted (revised) 10 September 1992
 I3C NMR ASSIGNMENTS FOR BRASSINOSTEROIDS 95

mode ('H nuclei detection) using the following pulse

sequences and parameters : H detected heteronuclear
R1 &R3
2 ' lq
a \%-
27
multiple quantum coherence (HMQC) spectroscopy,
'H90"-A-' 3C90"-t, /2- 'H 180"-t,/2- 13C90"-Acq-PD
(Acq = 0.387 s, P D = 1.7 s, A = 3.75 ms); 'H detected
R2 3 ~

b '.- heteronuclear multiple bond connectivity (HMBC)

0
spectroscopy, 'H90"-A1-' 3C90"-A,-' 3C900-t '/2-
CA R1 : a-OH, R 2 : a-OH, R3: a C
'H180"-t,/2-'3C 9W-Acq-PD (Acq = 0.387 S,
TY R ' : H, R 2 : a-OH, R 3 : a P D = 2.0 s, A, = 60 ms, A2 = 3.75 ms). Homonuclear
TE R1 : H, R2 : p-OH, R3: a correlation spectra (absolute data) were acquired with a
EC R': a-OH, R 2 : a-OH, R3: b spectral width of 1400 Hz using a 1024 x 256 data set,
HC R' : a-OH, R2 : a-OH, R3: c zero filling to 1024 x 512 and transforming after multi-
DS R1 : a-OH, R 2 : a-OH, R3: d plication by a sine-bell function. Heteronuclear corre-
HD R': a-OH, R2: a-OH, R3: e lation spectra (absolute data) were acquired with
spectral widths of 5700 (13C) and 1400 Hz ('H) using a
2048 x 128 data set, zero filling to 2048 x 256 and
transforming after multiplication by an exponential

&
d R 3 R3 function. Exceptionally large spectral widths of 11 O(N)
'
( 3C) and 1700 Hz ('H) were used for DS and HD.
R'
R2 R,
R* RESULTS
0 0

BR R' : a-OH, R 2 : a-OH, R 3 : a 1 R' : a-OH, R 2 : a-OH, R 3 : a

2 R' : H. R 2 : a-OH, R3: a 3 R 1 : H,
4 R1 : H, R 2 : p-OH, R3: a 5 R ' : H,
Fiaure 1. Structure of eight natural-type brassinosteroids
[brassinolide (BR), castasterine (CA), typhasterol (TY). teas-
terone (TE), 24-epicastasterone (EC), homocastasterone (HC),
dolichosterone (DS), and homodolichosterone (HD)] and five
synthetic derivatives 1-5, showing the numbering system used.
NMR spectroscopy
'H and 13C NMR spectra were recorded on a JEOL
GX 270 Fourier transform NMR spectrometer at 270
and 67.8 MHz, respectively, using a probe for 5 mm
tubes. Each compound was dissolved in a mixture of
chloroform-d and methanol-d, (4 :1, v/v) containing
tetramethylsilane as internal standard. The deuterium
signal from chloroform-d was used for the field-
frequency lock. 'H signals were accumulated with a 6.1
ps pulse width (45"), 2.73 s acquisition time (Acq) and
1.27 s pulse delay (PD) for 16384 data points. I3C
signals were accumulated with a 4.2 ps pulse width
(45"), 0.963 s Acq and the following P D for 32768 data
points: 1.7 s for a complete decoupling experiment and
for a DEPT experiment with a 'H 135" pulse in the
decoupling mode (DEPT 135"), and 1.2 s for the off-
resonance decoupling experiment. Two-dimensional
(2D) NMR spectra of each compound were also mea-
sured with the same spectrometer using the following
pulse sequences and parameters: 'H-'H correlation
spectroscopy (COSY), 'H90"-tI-'H900-Acq-PD
(Acq = 0.366 s, PD = 1.27 s); long-range COSY, 'H
90"-A-tl-'H 90O-A-Acq-PD (Acq = 0.364 S, P D = 2.0
s, A = 0.2 s); 13C-'H shift correlation spectroscopy with
13C nuclei detection (C-H COSY), 1H90"-t1/2-'3C
180"-t 1/2-A1-' H90°, 3C900-A2-' HBBD/Acq-PD
(Acq = 0.18 s, P D = 1.5 s, A1 = 3.3 ms, A2 = 1.65 ms);
long-range C-H COSY (Acq = 0.180 s, P D = 1.7 s,
A1 = 33.3 and 65.0 ms, A2 = A1/2). I3C-'H shift corre-
lation spectra of 4 and 5 were analysed by the inverse
R 2 : a-OH, R3: a
'H and I3C NMR signal assignments for brassinolide
R2: @-OH,R3: a
(BR) and castasterone (CA)
BR and CA are easily differentiated by their character-
istic H-5 and H-7 signals. For BR the H-5 signal is at
3.15 ppm (dd) and that of H-7 (2H) at 4.11 ppm (d),
while in CA H-5 resonated at 2.71 ppm (dd) and one of
the H-7 pair, not overlapping with any other signals, at
2.28 ppm (dd). Starting from H-5 in each case, corre-
lation peaks in the respective COSY spectra revealed
the signals for H-4, H-3, H-2 and H-1. However, precise
chemical shift values for H-1 and H-4 were not obtained
because they are masked by other signals. H-2 and H-3
could thus be distinguished from the other carbinol
methine protons, H-22 and H-23. Since these two brass-
inosteroids have the same side-chain which is remote
from the variations in the skeletal structure, it is
expected that the two sets of protons for H-22 and H-23
will have almost the same chemical shifts, and this
proved to be the case. The COSY spectra of BR and CA
also established the assignments for H-8 which shows a
correlation peak with H-7, and for the side-chain
protons H-20 to H-28 which show a series of corre-
lation peaks analysed from H-22 and H-23. However,
some difficulty was encountered since the resonances for
H-8, H-20, H-24 and H-25, which appear in the compli-
cated methylene and methine proton region of the
spectra, could not be observed as distinct lines in the
1D spectra. Each steroid has two angular methyl
groups H-18 and H-19 which resonate as sharp singlets;
H-19 can be differentiated from H-18 by its correlation
peak with H-5 in the long-range COSY spectra. Our
assignments of the angular methyl signals of BR and
CA agree with the results of Baba et aL6
The chemical shifts of the carbonyl carbon (C-6) in
BR and CA are distinct and characteristic. With the
proton signals assigned unambiguously, the four car-
binol carbons (C-2, C-3, C-22 and C-231, the two
carbons (C-5 and C-7) adjacent to either the carbonyl
or the lactone group, and six methyl carbons (C-18,
96 T. ANDO, M. ABURATANI, N. KOSEKI, S. ASAKAWA, T. MOURI AND H. ABE

Table 1. 'H and " C NMR data for brassinolide (BR) and castasterone (CA)
' H NMA ' j C NMA
chemical shifts' chemical shifts Correlation
(wm) (ppm) in long-range
Position BR CA BR CA MultiplicityQ C-H caw
1 -1.55, -1.85 -1.6, rb1.75 41.3 40.0 t H-19
2 3.62 m -3.7 68.0 68.2 d
3
4
5
- 3.96 bs
1.9, -2.05
3.15 ddd
3.99 bs
-1.7, -1.85
2.71 dd"
68.0
31.4
41.2
68.4
26.5
51.1
d
t
d H-19
6 177.6 213.9 S
7 4.11 d' -2.05, 2.28 dd' 70.8 46.9 t
8
9
10
2.1.75
-1.3 -
-1.8
1.45
39.3
58.3
38.4
38.1
53.9
42.9
d
d
S
H-19
H-19
11 -1.4. -1.8 -1.35, -1.7 22.4 21.4 t

-
12 -1.2, 2.0 -1.25, -2.05 39.8 39.7 t H-18
13 42.6 43.0 S H-18
14 -1.2 1.35 51.4 56.8 d H-18
15 -1.2, ~ 1 . 7 -1.15, -1.6 24.8 24.0 t
16 -1.3, -2.0 -1.3, -2.0 27.7 27.7 t
17 -1.55 -1.65 52.4 52.5 d H-18, H-21
18 0.73 s 0.70 s 11.8 12.0 q
19 0.91 s 0.76 s 15.5 13.6 9
20 X1.45 ~ 1 . 5 37.1 37.1 d H-21
21 0.89 dh 0.90 d ' 11.9 12.1 q
22 3.51 dJ 3.52 d' 74.5 74.6 d H-21
23 3.68 d' 3.69 dJ 73.3 73.4 d H-28
24 ~1.15 -1.2 40.4 40.5 d H-26, H-27, H-28
25 -1.6 -1.65 30.8 30.8 d H-26, H-27, H-28
26' 0.94 d' 0.94 dk 20.9 20.8 q
27' 0.96 d ' 0.97 dk 20.8 21 .o q
28 0.84 dh 0.84 dh 10.2 10.3 q
a Methylene and methine protons absorbed at 1.I 5-2.05 ppm and their overlap is such that their individ-
ual multiplicity is not clear. The chemical shifts were estimated from the correlation peaks in COSY and
C-H COSY spectra.
bThe 13C off-resonance decoupling experiments were confirmed by DEPT 135" experiments.
Correlation peaks were observed in long-range C-H COSY spectra measured with a A, of either 33.3 or
65.0 ms.
J = 12.4.5 Hz.
J = 12.5, 3.5 Hz.
'J=SHz.
J = 13, 4.5 Hz.
hJ= 7 Hz.
'J=6Hz.
'J=8Hz.
J = 6.5 Hz.
' Assignments for these two centers are not unambiguous and may be reversed.

C-19, C-21, C-26, C-27 and C-28) of both steroids could
be readily assigned by C-H COSY spectra. Although
the COSY spectra indicated the chemical shifts of the
six protons H-1, H-4, H-8, H-20, H-24 and H-25, they
are buried in other methylene and methine proton
signals and therefore the assignments of the correspond-
ing carbons (C-1, C-4, C-8, C-20, C-24 and C-25) from
only the C-H COSY experiments were still incomplete.
To remove this uncertainty we measured the long-
range C-H COSY spectra, and these also provided the
assignments of some other carbon signals (C-9-C-17).
Thus, H- 19 shows correlation peaks with four carbons,
one of which is confirmed as the already assigned C-5.
The others are established as the methylene, methine
and quaternary carbons C-1, C-9 and C-10, respectively.
The correlation peaks with H-18 revealed the methy-
lene, methine and quaternary carbons C-12, C-14 and
C-13, respectively. Another correlated methine carbon
was identified as C-17, since it also shows a correlation
peak with H-21 and was thereby distinguished from
C-14. Furthermore, correlation peaks with four methyl
protons (H-21, H-26, H-27 and H-28) confirmed the 13C
signal assignments for the side-chain. Long-range C-H
COSY spectra also show correlation peaks between
C-5-H-3, C-9-H-7, C- 10-H-5, C-21-H-22 and C-28-H-
23, and these data also support the above assignments.
The chemical shifts of three other methylene carbons
(C-11, C-15 and C-16), which did not show any corre-
lation with methyl protons or carbinol methine protons
even in the long-range C-H COSY spectra, were assign-
ed based on the data of other The chemical
shifts of the corresponding attached protons were esti-
mated conversely by analysing the correlation peaks of
C-9, C-11, C-12 and C-14-C-17 in the C-H COSY
spectra. The complete NMR data for BR and CA are
presented in Table 1.
 13C N M R ASSIGNMENTS F O R BRASSINOSTEROIDS 97

Table 2. 'H and I3C NMR assignments of typhasterol (TY), teasterone (TE), their Bhomo-
oxa derivatives 2,3,4 and 5, and the castasterone-B-homo-6-oxaderivative 1
3a-OH steroids 38-OH steroids Za,3a-(OH), steroid
TY 2 3 TE 4 5 1
Position 'ti NMR chemical shifts (ppm)

2 ?r 1.65" -1.6 -1.6 ~ 1 . 4 5 ~ -1.3 -1.3 -3.7

-1.85b -1.8 -1.75
3 4.09 24.1 4.14 3.52 -3.5 -3.55 3.97
4 -1.7= -1.75 -1.8 -1.4b -1.9 -1.7 2.1.9
-2.1 -2.0 ~ 1 . 8 ~ -2.15 -2.15
5 2.74 3.22 4.66 2.24 2.87 4.27 4.64
7 -2.05 4.15 2.49 22.0 4.02 2.42 2.49
2.28 2.31 4.12 2.53
'%NMR chemical shifts (ppm)

1 31.9 33.1 31.4 36.8 39.0 35.2 39.2

2 27.9" 28.0 27.7 29.6d 31 .O 30.4 67.8
3 64.9 64.4 65.8 70.2 69.3 68.2 68.8
4 27.6" 32.5 35.7 30.4d 35.2 38.1 34.3
5 52.0 41.9 80.5 57.0 46.9 81.8 79.3
6 21 4.6 178.3 176.7 212.6 176.1 175.1 176.2
7 46.9 70.8 38.1 46.8 70.7 38.2 38.0
8 38.4 39.7 35.2 38.3 39.5 34.9 34.6
9 53.9 58.4 58.1 54.0 58.4 58.0 57.8
10 41.9 36.4 39.9 41.3 36.1 39.3 40.6
11 21.3 22.4 22.4 21.7 22.8 22.8 22.4
12 39.7 39.9 39.9 39.7 39.8 39.8 39.8
13 43.0 42.6 42.7 42.9 42.5 42.5 42.6
14 56.9 51.6 55.6 56.8 51.4 55.5 55.4
15 24.0 24.9 25.4 24.0 24.8 25.2 25.3
16 27.7 27.7 27.3 27.7 27.6 27.2 27.3
17 52.5 52.5 52.8 52.5 52.3 52.6 52.7
18 12.0 11.9 11.9 12.0 11.9 11.8 11.8
19 12.4 14.6 11.6 13.2 15.1 12.3 12.6
a-d Assignments of the pairs of resonances indicated are not unambiguous and may be reversed.

Table 3. Differences in the I3C NMR chemical shifts (ppm) between. keto-
brassinosteroid and their Bhomo-oxa (lactone) derivatives
Differences for a 7-oxa derivative Differences for a 6-oxa derivative
Positlon A(CA - B A ) A(TY - 2) A(TE - 4) A(CA - 1 ) A(TY - 3) A(TE - 5)

1 -1.3" -1.2 -2.2 0.8 0.5 1.6

2 0.2 -0.1 -1.4 0.4 0.2 -0.8
3 0.4 0.5 0.9 -0.4 -0.9 2.0
4 -4.9 -4.9 -4.8 -7.8 -8.1 -7.7
5 9.9 10.1 10.1 -28.2 -28.5 -24.8
6 36.3 36.3 36.5 37.7 37.9 37.5
7 -23.9 -23.9 -23.9 8.9 8.8 8.6
8 -1.2 -1.5 -1.2 3.5 3.2 3.4
9 -4.4 -4.5 -4.4 -3.9 -4.2 -4.0
10 4.5 5.5 5.2 2.3 2.0 2.0
11 -1 .o -1 .I -1.1 -1.0 -1.1 -0.9
12 -0.1 0.0 -0.1 -0.1 -0.2 -0.1
13 0.4 0.4 0.4 0.4 0.3 0.4
14 5.4 5.3 5.4 1.4 1.3 1.3
15 -0.8 -0.9 -0.8 -1.3 -1.4 -1.2
16 0.0 0.0 0.1 0.4 0.4 0.5
17 0.1 0.0 0.2 -0.2 -0.3 -0.1
18 0.2 0.1 0.1 0.2 0.1 0.2
19 -1.9 -2.2 -1.9 1 .o 0.8 0.9
=This value was obtained by subtracting the chemical shift (40.0ppm) for C-1 in
CA from the chemical shift (41.3ppm) for C-1 in BR.
98 T. ANDO, M. ABURATANI., N. KOSEKI, S. ASAKAWA, T. MOURI AND H. ABE

Table 4. 'H and 13C NMR data for 24-epicastasterone (EC), might be caused by the inverse configuration of the 3-
homocastasterone (HC), dolichosterone (DS) and hydroxy group in TE. The amounts of 4 and 5 available
homodolichosterone (HD) were insufficient for 13C signal analyses by the usual 2D
experiments detecting I3C nuclei. However, their 3C
EC HC 0s HD
Position 'H NMR chemical shifts (ppm)
assignments were confirmed by measuring HMQC and
HMBC spectra (detecting 'H nuclei), which correspond
21 0.97 0.91 0.94 0.92
3.67 3.56 3.60 3.65
to the C-H COSY and long-range C-H COSY spectra,
22
23 3.37 3.69 4.00 3.92 respectively.
24
25
26"
-1.45
xl.9
0.86
-1.05
-1.85
0.95
-

2.26
1.09
--

2.7
1.07 13C NMR signal assignments for the side-chains in four
27" 0.91 0.97 1.11 1.14 analogues of castasterone
28 0.84 1.3,-1.45 5.02,5.04 5.48
29 - 0.94 - 1.71
Four CA analogues (EC, HC, DS and HD) were
'3CNMR chemical shifts (ppm) analysed by the above 2D NMR techniques, and their
17 52.7 52.7 52.9 53.0 'H and 13C NMR assignments are presented in Table 4.
20 40.3 37.2 37.0 36.7 Since their ring skeletons are identical with that of CA,
21 12.4 12.0 12.6 12.6 each 13C signal of the ring moiety of the molecules
22 72.5 74.5 75.7 75.1 appeared within f0.2 ppm of the chemical shift
23 76.0 72.6 76.6 74.8 observed in CA, except for C-17, which varied by up to
24 41.6 46.7 156.5 145.3 +0.5 ppm. The 13C chemical shifts of their side-chains
25 27.0 29.2 31 .O 27.7 are different to each other and reflect the structural
26 17.3 19.6 23.0 21.4
22.2 21.3 23.3 21.6
modifications at the 24-position. It is noteworthy that
27
28 10.9 19.1 1 10.6 123.1 CA and its (24s)-diastereomer (EC) showed different
29 - 13.8 - 13.7 3C spectra. Stereoselective synthesis directed towards
the configuration of the C-28 methyl group can there-
a.bAssignrnentsfor these two centres are not unambiguous and fore be easily confirmed by the 13C NMR measurement.
may be reversed.

~~ ~

13C NMR signal assignments for typhasterol (TY),
teasterone (TE) and their B-homo-oxa derivatives
The 13C NMR spectra of TY, TE, 1, 2 and 3 were
analysed (Table 2) using the same 2D NMR techniques
as described above for the BR and CA assignments.
These steroids have the identical side-chain (a in Fig. 1)
and show a set of similar signals for C-20 to C-28. Each
13C signal of the side-chain is within f0.2 ppm of the
chemical shifts observed in BR. Since T Y and TE lack
the 2-hydroxy group, the chemical shift difference
between H-2 and H-4 is now small (see Table 2), and it
was difficult to differentiate between these protons even
with the aid of the H-4-H-5 correlation peaks in the
COSY spectra. Thus, the C-2 and C-4 signal assign-
ments are still tentative. In 2 and 3, B-homo-oxa deriv-
atives of TY, H-4 resonated at lower field than H-2
because of the B-ring modification (see Table 2). In this
case H-2 and H-4 were easily distinguished and the C-2
and C-4 assignments are unambiguous.
The B-ring modification in brassinosteroids affected
the I3C chemical shifts of not only C-6 but of almost all
carbons of the ring system (A to D). The differences in
the shift values between a keto-brassinosteroid and the
corresponding B-homo-7-oxa or 6-oxa derivatives are
presented in Table 3. The differences between CA and
BR [A(CA - BR)] have a high degree of coincidence
with those between T Y and 2 [A(TY-2)]. The values of
A(CA-1) and A(TY-3) are also similar. The "C signal
assignments for 4 and 5, B-homo-oxa derivatives of TE,
were tentatively assigned as shown in Table 2 based on
the differences in the shift values. The difference values
show variations for some carbons in the A-ring. This
DISCUSSION
Brassinosteroids are novel steroids ; this particularly
applies to BR, which contains the homo-6-keto-7-oxa
grouping in the B-ring. This compound can be synthe-
sized via a Baeyer-Villiger reaction of the 6-keto com-
pound (CA), a reaction which also produces the 6-oxa
isomer as a by-product. Brassinosteroids do not have
any chromophores suitable for HPLC analysis with a
UV detector, and for GC analysis have to be derivatized
to enhance their volatility. Because of these limitations
13C NMR is one of the most useful techniques for
determining the purity of BR and other brass-.
inosteroids.
Steroids show a complex pattern of 'H signals
around 1-2 ppm, precluding direct assignments of indi-
vidual resonances, and analyses of the 13C NMR
spectra are not easily performed by C-H COSY mea-
surements. Brassinosteroids, however, have two angular
methyl groups, at the 10- and 13-positions, and a
further three or four methyl groups in the side-chain,
and these serve as useful starting points in interpreting
NMR correlation experiments. On using C-H COSY
measurements and emphasizing the long-range coug-
ling, several informative correlation peaks were
observed between these methyl protons and the carbons
connected with the protons through two or three bonds.
A strong correlation peak with the methyl protons is
usually observed in a long-range C-H COSY spectrum
and is useful for I3C signal assignment, as we have
already reported for other corn pound^.^^'^ Correlation
peaks with methine or methylene protons in the spectra
are weak, but they are still valuable in reaffirming the
 I3C NMR ASSIGNMENTS FOR BRASSINOSTEROIDS
signal assignments proposed for complex compounds
such as brassinosteroids. Thus, the 3C assignment for
each brassinosteroid, except for the C-11, C-15 and
C-16 signals, was accomplished without referring to any
published data.
Natural brassinosteroids isolated to date show a
variety of structural modifications in the A- and B-rings,
and in the side-chains, and it is plausible that new com-
pounds remain to be identified which incorporate many
of these differences. The NMR data presented in this
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99
study for 13 brassinosteroids therefore provide a basis
for NMR analyses and, hence, structural assignments
for new compounds of this class as they are isolated,
synthesized or structurally modified.
Ackn,-,wl,,dge,,,ent
We thank Dr R. F. Toia of the University of California, Berkeley, CA,
for kindly reading the manuscript.
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