MAGNETIC RESONANCE IN CHEMISTRY

Magn. Reson. Chem. 37, 856–859 (1999)

Note

NMR assignments of ellagic acid derivatives

Xing-Cong Li,1 Hala N. Elsohly,1 Charles D. Hufford,1,2 and Alice M. Clark1,2 ∗

1
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of Mississippi, University,
Mississippi 38677, USA
2
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, USA

Received 2 March 1999; revised 25 May 1999; accepted 25 May 1999

ABSTRACT: HMBC spectroscopy optimized for small couplings was employed to determine the four-bond and two-
bond proton carbon correlations on the aromatic rings of ellagic acid derivatives. Complete 13 C NMR assignments
of 30 -O-methyl-3,4-methylenedioxyellagic acid 40 -O-ˇ-D-glucopyranoside (1), 3,30 -di-O-methylellagic acid 40 -O-ˇ-D-
xylopyranoside (2), 3,30 ,4-tri-O-methylellagic acid 40 -O-ˇ-D-glucopyranoside (3) and ellagic acid (4) were achieved
using this technique. This study indicates that optimization of the delay time in the HMBC spectrum is crucial in
assigning the 13 C NMR signals of phenolic compounds with highly oxygenated quaternary carbons. Copyright  1999
John Wiley & Sons, Ltd.
13
KEYWORDS: NMR; C NMR; HMBC; small coupling; four-bond and two-bond proton carbon correlations; ellagic
acid derivatives

INTRODUCTION
Ellagic acid derivatives are widely distributed in plant
kingdom.1 Since 12 non-protonated and only two proto-
nated aromatic carbons are present in these compounds,
the assignment of the quaternary carbon signals with
close chemical shifts is a significant challenge. Only in
recent years, with the advent of modern NMR tech-
niques such as selective INEPT,2 COLOC3 and het-
eronuclear multiple bond connectivity (HMBC),4,5 has it
become practically possible to assign complicated qua-
ternary carbon signals within one molecule. Over the
past decade HMBC has been demonstrated as the most
powerful long-range H/C correlation spectroscopy in nat-
ural products chemistry. This technique optimized for
small couplings was employed to assign unambiguously separated by only a few tenths of a ppm. For example,
the 13 C NMR signals of several ellagic acid deriva- five quaternary carbon signals appear in the region of υ
tives: 30 -O-methyl-3,4-methylenedioxyellagic acid 40 -O- 111–115 (υ 111.2, 111.8, 112.3, 112.9 and 114.8). To
ˇ-D-glucopyranoside (1), 3, 30 -di-O-methylellagic acid 40 - assign unequivocally the carbon signals of 1, the fol-
O-ˇ-D-xylopyranoside (2), 3, 30 ,4-tri-O-methylellagic acid lowing experiments were performed. The HMQC was
40 -O-ˇ-D-glucopyranoside (3) and ellagic acid (4), which first employed to determine the direct H/C correlations
were isolated from Nyssa sylvatica and Miconia myriantha and assign protonated carbons. With the aid of COSY,
and whose NMR assignments were incomplete and inac- the 1 H and 13 C NMR signals of the sugar moiety were
curate in the literature.6,7,8 also assigned (Tables 1 and 2). HMBC was then per-
formed to establish the long range H/C correlations of
the aromatic rings. In the first HMBC experiment, the
RESULTS AND DISCUSSION delay time was set to 50 ms, corresponding to a J(C,H)
Compound 1 displayed 22 distinct carbon signals in the value of 10 Hz. In addition to the correlations originating
13
C NMR spectrum, but some of these signals were from the methoxy, methylenedioxy and sugar, the impor-
tant correlations between each aromatic proton and four
* Correspondence to: A. M. Clark, National Center for Natural Products quaternary carbons were revealed (Fig. 1). Based on this
Research, Research Institute of Pharmaceutical Sciences, University of experiment, four pairs of quaternary carbon signals were
Mississippi, University, Mississippi 38677, USA. assigned: C-1/C-10 , C-3/C-30 , C-4/C-40 and C-7/C-70 . The
E-mail: ncnpr@olemiss.edu
Contract/grant sponsor: National Institute of Allergy and Infectious remaining two pairs of quaternary carbons, C-6/C-60 and
Diseases; Contract/grant number: AI 27094. C-2/C-20 , which had small couplings with the aromatic

Copyright  1999 John Wiley & Sons, Ltd. CCC 0749–1581/99/110856–04 $17.50
 NMR ASSIGNMENTS OF ELLAGIC ACID DERIVATIVES 857

13
Table 1. C NMR data for compounds 1–4 in DMSO-d6 (υ ppm)

C 1 2 2a 3 4 4a 4b
1 114.8 111.0 110.67 112.4 112.2 106.8 112.3
2 130.8 141.5 140.60 140.8 136.3 136.2 136.4
3 138.0 140.1 140.00 140.7 139.4 140.4 140.7
4 150.3 152.7 152.60 154.1 148.0 148.2 153.0
5 103.7 111.4 111.41 107.3 110.2 109.8 111.4
6 111.2 112.7 112.27 112.2 107.6 112.4 107.7
7 157.6 158.3 158.10 158.1 159.0 159.1 159.2
10 112.9 114.1 113.85 113.8
20 140.8 140.8 141.18 140.9
30 141.7 141.8 141.85 141.6
40 151.9 151.1 151.05 151.8
50 112.3 111.8 112.00 112.0
60 111.8 111.8 112.27 112.0
70 157.0 158.2 158.20 157.9
—CH2 — 104.5
3-OMe 60.9 60.80 61.2
4-OMe 56.6
30 -OMe 61.6 61.5 61.50 61.6
Sugar:
100 101.4 101.6 101.98 101.2
200 73.2 72.9 72.92 73.2
300 76.4 76.0 75.98 76.4
400 69.4 69.1 69.98 69.4
500 77.2 65.7 65.67 77.2
600 60.5 60.5
a
Data from Ref. 2.
b
Data from Ref. 4.

Table 2. 1 H NMR data for compounds 1–4 in DMSO-d6 [υH (ppm) with J.H2 / in parentheses

1 2 3 4
H-5 7.29 (br d, 2.4) 7.52 (s) 7.50 (s) 7.47 (s)
H-50 7.75 (s) 7.74 (s) 7.80 (s) 7.47 (s)
—CH2 — 6.34 (s)
3-OMe 4.03 (s) 4.04 (s)
4-OMe 4.06 (s) 3.97 (s)
30 -OMe 4.11 (s) 4.11 (s)
H-100 5.14 (d, 6.7) 5.14 (d, 7.2) 5.17 (d, 6.3)
H-200 3.40 3.33 3.45
H-300 3.40 3.32 3.44
H-400 3.25 3.38 3.26
H-500 a 3.50 3.82 (dd, 10.0, 4.0) 3.47
H-500 b 3.33
H-600 a 3.75 (br d, 11.0) 3.73 (br d, 10.0)
H-600 b 3.56 (dd, 11.6, 5.1) 3.54 (dd, 11.2, 5.0)

The second HMBC experiment was therefore set to a

Figure 1. Key HMBC correlations for 1 ( D 50 ms).
protons (1–2 Hz),9 were unassignable owing to the lack of
HMBC correlations under the conditions described above.
delay time of 400 ms, corresponding to a J(C,H) value
of 1.25 Hz. Additional cross peaks were observed in this
experiment (Fig. 2). The aromatic proton signal at υ 7.29
displayed cross peaks with C-2 at υ 130.8 and C-6 at υ
111.2. The former was a 4 J(C,H) correlation, while the
latter was a 2 J(C,H) correlation in which the correlated
carbon was a non-oxygenated quaternary carbon. The aro-
matic proton signal at υ 7.75 showed two 4 J(C,H) corre-
lations with C-20 at υ 140.8 and C-1 at υ 114.8. From the
above two experiments, all of the direct correlated carbon
information except that of C-60 were obtained. Therefore,

Copyright  1999 John Wiley & Sons, Ltd. Magn. Reson. Chem. 37, 856–859 (1999)
858 X.-C. LI ET AL.
Figure 2. Key HMBC correlations for 1 ( D 400 ms).
Figure 3. Key HMBC correlations for 1 ( D 200 ms).
the remaining carbon signal at υ 111.8 could be assigned
to C-60 .
Although the two experiments allowed complete assign-
ments of the quaternary carbons, a third HMBC experi-
ment with a delay time of 200 ms, corresponding to an
n
J(C,H) value of 2.5 Hz, was also performed. More cross
peaks were observed in this experiment (Fig. 3). Each aro-
matic proton signal correlated with seven carbon signals
covering all the 2 J(C,H), 3 J(C,H) and 4 J(C,H) correla-
tions. In addition, this experiment was much more sensi-
tive than the previous one in which a longer delay time
(400 ms) was used.
The above three HMBC experiments led to the con-
clusion that combination of the first and the third HMBC
experiments could readily achieve the complete 13 C NMR
assignments of 1. This methodology was then applied to
the assignments of 2–4, which had been questioned in the
literature.7,8
First the HMBC spectrum with a delay time of 50 ms
[to determine the 3 J(C,H) and ortho-oxygenated 2 J(C,H)
correlations] was performed, then a delay time of 200 ms
was used to obtain the correlations, including the 4 J(C,H)
and non-ortho-oxygenated 2 J(C,H). The 13 C and 1 H NMR
assignments of these compounds are presented in Tables 1
and 2. The assignments of the aglycone moiety of 2
were in accordance with that of 3, 30 -di-O-methylellagic
acid 40 -O-ˇ-D-glucopyranoside, which were made by
a combination of selective decoupling and HMBC
techniques.9
This study indicates that HMBC spectroscopy opti-
mized for small couplings, can be effectively used to
assign the signals of a variety of ellagic acid derivatives,
and also related phenolic compounds with highly oxy-
genated quaternary carbons. The delay time,  D 200 ms
[corresponding to an n J(C,H) coupling of 2.5 Hz], is a
compromise value which can intensify small coupling
Copyright  1999 John Wiley & Sons, Ltd.
correlations such as 4 J(C,H) and 2 J(C,H) on the aromatic
rings of phenolic compounds.
EXPERIMENTAL
Isolation of compounds 1 and 3
The dried powdered wood (1.1 kg) of Nyssa sylvatica Marsh (Nys-
saceae) (collected in Mississippi, voucher specimen BUR 240693
deposited at NCDNP) was extracted with 95% EtOH (2.8 l ð 3) at 37 ° C
for 3 h. Removal of the solvent at 45 ° C yielded an EtOH extract (52.5 g).
The EtOH extract (50.1 g) was suspended in H2 O (1.6 l) and extracted
successively with CHCl3 (1 l ð 3), EtOAc .1 l ð 3/ and 1-BuOH (satu-
rated with H2 O, 1 l ð 3). The combined 1-BuOH layers were evaporated
to dryness in vacuo .45 ° C/ to give a yellow residue (19.24 g). This
residue (18 g) was subjected to column chromatography on silica gel
using CHCl3 –MeOH–H2 O (70 : 10 : 1 to 30 : 10 : 1) as the eluting sol-
vent (7.5 l), followed by MeOH (1.5 l). Fractions of 60 ml each were
collected. Compound 3 was crystallized from fractions 26–30 as granu-
lar crystals (7.0 mg). Identification of 3 was made by comparison of its
UV, IR and 1 H NMR spectra (Table 2) with reported data.1 Compound 1
was crystallized from fractions 61–90 as granular crystals (22.3 mg): 1 H
NMR (pyridine-d5 , 300 MHz), υ 8.46 (1H, s, H-50 ), 7.75 (1H, s, H-5),
6.35 (2H, dd, J D 3.4, 0.9 Hz, OCH2 O), 5.91 (1H, dd, J D 5.3, 1.9 Hz,
Glc H-100 ), 4.61 (1H, dd, J D 11.9, 1.9 Hz, Glc H-600 a), 4.46–4.16 (5H,
m), 4.26 (3H, s, 30 -OMe); 13 C NMR (pyridine-d5 , 75 MHz), υ 158.4,
157.7, 152.8, 151.0, 142.6, 141.7, 138.7, 131.8, 115.6, 113.7, 113.3,
112.8, 112.3, 104.7, 104.4, 102.7, 78.7, 78.1, 74.5, 70.8, 61.9, 61.7.
Identification of this compound was made by comparison of its UV, IR,
and 1 H NMR spectra with those reported previously.6
Isolation of compounds 2 and 4
The dried powdered twig-leaf (170 g) of Miconia myriantha Bentham
(Melastomataceae) (collected in Peru, voucher specimen IBE 9732
deposited at NCDNP) was extracted with 95% EtOH .1.2 l ð 3/ at
37 ° C for 3 h. Removal of the solvent at 45 ° C yielded an EtOH extract
(10.5 g). The EtOH extract (5.0 g) was subjected to column chromatog-
raphy on silica gel using a step gradient mobile phase consisting
of CHCl3 –MeOH with increasing amounts of MeOH (9 l), followed
by MeOH (1.5 l). Similar fractions were pooled according to TLC
to yield a total of 17 fractions (fractions 1–17). Rechromatography
of the combined fractions 15 and 16 on a silica gel column using
CHCl3 –MeOH–H2 O (70 : 10 : 1) as the mobile phase afforded 2 as gran-
ular crystals (6.9 mg). Identification of this compound was made by
comparison of its 1 H and 13 C NMR with those reported previously.7
Rechromatography of fraction 17 on a Diaion HP-20 column using
aqueous MeOH as eluent yielded 4 as a pale yellow powder (11.3 mg).
Identification of 4 as ellagic acid was made by a direct comparison of its
TLC behavior and 1 H and 13 C NMR spectra with those of an authentic
sample from Sigma Chemical Co (St Louis, MO, USA).
NMR spectra
NMR spectra were obtained at ambient temperature on
either a Bruker Avance DPX-300 spectrometer operat-
ing at 300 MHz for 1 H and 75 MHz for 13 C (1 H 90°
pulse width D 11 µs, 13 C 90° pulse width D 5.5 µs)
or a Bruker Avance DRX-400 spectrometer operating
at 400 MHz for 1 H and 100 MHz for 13 C (1 H 90° pulse
width D 14.25 µs, 13 C 90° pulse width D 8.0 µs). Sam-
ples were dissolved in DMSO-d6 at a concentration of
20–50 mg ml 1 . Chemical shifts (υ, ppm) are relative
to internal TMS. One-dimensional 1 H and 13 C spectra
were recorded for a 6188 Hz spectral width with 32 768
data points and a 13 550 Hz spectral width with 65 536
data points, respectively, on the DRX-300 spectrometer,
corresponding to 0.19 Hz 1 H resolution and 0.21 Hz 13 C
Magn. Reson. Chem. 37, 856–859 (1999)
 NMR ASSIGNMENTS OF ELLAGIC ACID DERIVATIVES
resolution, and for a 8250 Hz spectral width with 32 768
data points and a 22 124 Hz spectral width with 65 536
data points, respectively, on the DRX-400 spectrome-
ter, corresponding to 0.25 Hz 1 H resolution and 0.34 Hz
13
C resolution. Two-dimensional COSY and HMQC were
measured with the usual pulse programs and acquisition
parameters. HMBC experiments were carried out using
a standard pulse program as follows. For compound 1,
the experiments with  D 50 ms and  D 400 ms
were performed on the DRX-400 spectrometer. The for-
mer consisted of 2K ð 256 spectra with NS D 48 and
the latter consisted of 2K ð 256 spectra with NS D 64.
The experiment with  D 200 ms was run on the DRX-
300 spectrometer consisting of 2K ð 256 spectra with
NS D 96. For compounds 2–4, all the HMBC spectra
were obtained on the Bruker DPX-300 spectrometer con-
sisting of 2K ð 256 spectra with different NS. For 2, the
first experiment was with  D 50 ms and NS D 112 and
the second with  D 200 ms and NS D 96. For 3, the
first experiment was with  D 50 ms and NS D 96 and
the second with  D 200 ms and NS D 96. For 4, the
first experiment was with  D 50 ms and NS D 80 and
the second with  D 200 ms and NS D 80.
Copyright  1999 John Wiley & Sons, Ltd.
859
UV and IR spectra
UV spectra were measured on a Hewlett-Packard Model
8453 spectrometer and IR spectra on an ATI Mattson
Genesis Series FTIR spectrometer.
Acknowledgment
This work was supported by the National Institute of Allergy and
Infectious Diseases, DAIDS, NIH, Bethesda, MD, grant No. AI 27094.
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Magn. Reson. Chem. 37, 856–859 (1999)