Biotransformation of o- and p-aminobenzoic acids and N-acetyl p- aminobenzoic acid by cell suspension cultures of Solanum mammosum

Article abstract of DOI:10.1016/S0031-9422(99)00112-0

Some new biotransformation products, p-aminobenzoic acid 7-O-β-D- glucopyranosyl ester, N-acetyl p-aminobenzoic acid 7-Oβ-D-glucopyranosyl ester, o-aminobenzoic acid 7-O-β-D-(β-1,6-O-D-glucopyranosyl)glucopyranosyl ester and o-aminobenzoic acid 7-O-β-D-gl

Full text of DOI:10.1016/S0031-9422(99)00112-0

Phytochemistry 51 (1999) 615±620
Biotransformation of o- and p-aminobenzoic acids and N-acetyl
p-aminobenzoic acid by cell suspension cultures of Solanum
mammosum
Achmad Syahrania, Evi Ratnasaria, Gunawan Indrayantoa,*, Alistair L. Wilkinsb
aLaboratory of Pharmaceutical Biotechnology, Faculty of Pharmacy, Airlangga University, Jl. Dharmawangsa Dalam, Surabaya, 60268, Indonesia
bDepartment of Chemistry, School of Science and Technology, The University of Waikato, Private Bag 3105, Hamilton, New Zealand
Received 30 June 1998; received in revised form 9 December 1998; accepted 9 December 1998
Abstract
Some new biotransformation products, p-aminobenzoic acid 7-O-b-D-glucopyranosyl ester, N-acetyl p-aminobenzoic acid 7-O-
b-D-glucopyranosyl ester, o-aminobenzoic acid 7-O-b-D-(b-1,6-O-D-glucopyranosyl)glucopyranosyl ester and o-aminobenzoic acid
7-O-b-D-glucopyranosyl ester were isolated from cell suspension cultures of Solanum mammosum following administration of p-
aminobenzoic acid, N-acetyl p-aminobenzoic acid or o-aminobenzoic acid respectively. N-acetyl p-aminobenzoic acid and N-
formyl p-aminobenzoic acid were also identi®ed as cell suspension metabolites of p-aminobenzoic acid. # 1999 Elsevier Science
Ltd. All rights reserved.
Keywords: Solanum mammosum; Cell suspension culture; Biotransformation; p-Aminobenzoic acid 7-O-b-D-glucopyranosyl ester; N-acetyl p-ami-
nobenzoic acid 7-O-b-D-glucopyranosyl ester; N-acetyl p-aminobenzoic acid; N-formyl p-aminobenzoic acid; o-Aminobenzoic acid 7-O-b-D-gluco-
pyranosyl ester; o-Aminobenzoic acid 7-O-b-D-(b-1,6-O-D-glucopyranosyl)glucopyranosyl ester
1. Introduction
Ohashi (1986) have reported that some cell suspension
cultures (e.g., Lithospermum erythrorhizon, Gardenia
jasminoides ) were able to transform salicylate deriva-
tives, such as salicyl alcohol, into salicin and isosalicin.
Dombrowski and Alfermann (1992) and Dombrowski
(1993) have reported the transformation of salicyl
alcohol into salicin and isosalicin, salicylic acid into
salicylic acid 2-O-b-D-glucopyranoside and salicylic
acid 7-O-b-D-glucopyranosyl ester in the suspension
cultures of Salix matsudana. In previous papers
(Syahrani, Indrayanto, Sutarjadi, & Wilkins, 1997b;
Syahrani, Indrayanto, Wilkins & Sutarjadi, 1997a), we
have reported that cultured suspension cells of
Solanum mammosum were able to transform salicyla-
mide into salicylamide 2-O-b-D-glucopyranoside and
salicyl alcohol into salicyl alcohol 2-O-b-D-glucopyra-
noside.
Biological transformation (biotransformation) using
plant cell suspension cultures serves as an important
tool in the structural modi®cation of compounds
possessing useful therapeutic activity (Kamel, Brazier,
Desmet, Flinaux, & Dubreuil, 1992). Various plant cell
cultures are capable of glycosylating a variety of ex-
ogenously supplied compounds (Suga & Hirata, 1990).
The glycosylation of simple phenols (e.g. salicyl alco-
hol and salicylic acid) by various cell suspension
cultures have been reported by Umetami, Tanaka, and
Tabata (1982). The glucosylation of isomeric hydroxy-
benzoic acids by cell suspension cultures of Mallotus
japonicus has been reported by Tanaka, Hayakawa,
Umetami, and Tabata (1990), while Mizukami, Terao,
Miura, and Ohashi (1983); Mizukami, Terao, and
We are now reporting the formation and structure
elucidation of p-aminobenzoic acid 7-O-b-D-glucopyra-
nosyl ester (1), N-acetyl p-aminobenzoic acid (2), N-
* Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00112-0

616
A. Syahrani et al. / Phytochemistry 51 (1999) 615±620
Table 1
13C NMR chemical shifts (d DMSO-d6) of some cell culture b-D-glu-
copyranosides and b-D-glucopyranosyl esters
formyl p-aminobenzoic acid (3) from exogenously sup-
plied p-aminobenzoic acid (7); N-acetyl p-aminoben-
zoic acid 7-O-b-D-glucopyranosyl ester (4) from N-
acetyl p-aminobenzoic acid (2); and o-aminobenzoic
acid 7-O-b-D-(b-1,6-O-D-glucopyranosyl) glucopyrano-
syl ester (5), o-aminobenzoic acid 7-O-b-D-glucopyra-
nosyl ester (6) from o-aminobenzoic acid (8) in
suspension cultures of S. mammosum.
Atom
1
4
5
6
1
115.1
131.6
112.7
153.9
112.7
131.6
164.6
132.2
130.7
118.2
144.0
118.2
130.7
164.2
169.0
24.1
107.9
151.8
116.5
134.5
114.6
131.0
166.0
108.1
151.8
116.5
134.5
114.7
131.0
166.1
2
3
4
5
6
7
2. Results and discussion
8
9
Toxicity of 2, 7 and 8 toward cell suspension cul-
tures of S. mammosum was investigated for concen-
trations in the range of 500±1500 mg l 1. Cell death
1'
2'
3'
4'
5'
6'
10
20
30
40
50
60
94.2
72.6
76.6
69.6
77.8
60.6
94.7
72.5
94.1
72.4
76.4
69.3
76.3
68.0
103.1
73.4
76.7
69.9
76.8
60.9
94.2
72.5
76.5
69.8
77.9
60.6
76.4
69.5
1
occurred at concentrations >1250 mg l for 2 and 7
1
and 1000 mg l for 8. At concentrations of 1000 mg
77.9
60.6
1
1
for (2) and (7) and 750 mg l for (8), cells sur-
l
vived, but did not grow (packed cell volumes Ð PCV
Ð remains relatively constant). Toxicity of 2 and 7 are
equivalent to the toxicity of salicylamide and salicyl
alcohol as previously reported (Syahrani et al., 1997b;
Syahrani et al., 1997a), whilst 8 appeared to be more
toxic toward this suspension cultures.
Incubation of cell suspension cultures of S. mammo-
sum Ð prepared, as reported previously (Syahrani et
al., 1997b; Syahrani et al., 1997a) Ð with p-aminoben-
zoic acid (7) followed by isolation, puri®cation by col-
umn chromatography and preparative TLC, gave
metabolite 1 (Rf 0.37) and mixture of metabolites 2
and 3 (Rf 0.51); incubation of N-acetyl p-aminobenzoic
acid (2) produced metabolite 4 (Rf 0.25). Metabolites 5
(Rf 0.08) and 6 (Rf 0.42) were obtained by incubation
of the cultures with o-aminobenzoic acid (8). Control
experiments showed that in the absence of cells, sub-
strates 7 (Rf 0.81), 2 (Rf 0.51) and 8 (Rf 0.82) remain
unchanged in the culture medium and that metabolites
1, 2, 3, 4, 5 or 6 were only produced when cell suspen-
sion cultures of S. mammosum were present in the
medium.
the glucopyranosyl unit was elucidated from a HMBC
correlation between H-1' (5.59 ppm) and C-7 (164.6
ppm), while the b-orientation of the glucoside linkage
was de®ned by the coupling constant (d, J=7.8 Hz)
determined for H-1' and by correlations between H-1',
and H-3' (3.25 ppm) and H-5' (3.39 ppm) respectively,
observed in the ROESY spectrum of 1.
One- and two-dimensional NMR analyses of a
minor fraction recovered after incubation with p-ami-
nobenzoic acid revealed it to be a ca 3:1 mixture of N-
acetyl p-aminobenzoic acid (2) and N-formyl p-amino-
benzoic acid (3). The HMBC spectrum of the mixture
showed correlations between the amide proton (-NH)
(10.09 ppm) of 2 and C-3 (=C-5) (117.5 ppm) and C-8
(168.2 ppm), and between H-2 (7.88 ppm) and C-7
(169.7 ppm), C-3 (117.5 ppm), C-4 (139.8 ppm) and C-
6 (129.5 ppm). Correlations were observed in the
ROESY spectrum between -NH proton of 2 and H-3
(=H-5) (7.57 ppm) and H-9 (2.12 ppm). The HMBC
spectrum of the mixture also included correlations
between the amide proton (-NH) (10.42 ppm) of 3 and
C-3 (=C-5) (117.7 ppm) and between the formyl pro-
ton (H-8) (8.34 ppm) of 3 and C-4 (138.7 ppm).
Correlations were also observed in the ROESY spec-
trum between the -NH proton of 3 and H-3 (7.57
ppm) and H-8 (8.34 ppm). A synthetic specimen of N-
acetyl p-aminobenzoic (2), subsequently utilized as a
substrate in a cell experiment, exhibited 1H and 13C
NMR resonances identical to those attributable to the
presence of 2 in the mixture of 2 and 3. GC/MS ana-
lyses of a methylated subsample of the mixture of 2
Incubation with p-aminobenzoic acid (7) a€orded
metabolite (1) which exhibited H and 13C NMR sig-
1
nals (Table 1) that indicated it was a glucosyl analogue
of (7). The positive ion electrospray mass spectrum
(ESMS) of 1, determined in a Na+ and NH4+ assisted
matrix, included pseudomolecular ions at m/z 300
(M+H)+, 317 (M+NH4)+ and 322 (M+Na)+, and
cluster ions at m/z 616 (2 M+NH4)+ and 621 (2
M+Na)+. The negative ion ESMS, determined in a
Cl assisted matrix, included pseudomolecular ions at
m/z 298 (M±H) , 334 (M+35Cl) and 336 (M+37Cl) ,
and cluster ions at m/z 597 (2 M±H) , 633 (M+35Cl)
and 635 (M+37Cl) . One- and two-dimensional NMR
spectral data (including COSY, ROESY, HSQC and
HMBC spectra) identi®ed 1 as p-aminobenzoic acid 7-
O-b-D-glucopyranosyl ester. The site of attachment of

A. Syahrani et al. / Phytochemistry 51 (1999) 615±620
617
and 3 a€orded peaks assigned to methyl esters of 2
(compound 9) [m/z 193 (M+)] and methyl ester of 3
(compound 10) [m/z 179 (M+)], respectively.
M+Na)+ cluster ions (m/z 923 and 945 respectively),
while the negative ion ESMS of 5, determined in a Cl
assisted matrix, included ions identi®ed as (M±H) ,
(M+35Cl) and (M+37Cl) pseudomolecular ions
(m/z 460, 496 and 498 respectively).
The 13C NMR spectrum of metabolite 4 (Table 1),
isolated after incubation of N-acetyl p-aminobenzoic
acid (7) indicated it to be a glucopyranosyl analogue
of 7. The 1H NMR spectrum of the metabolite
included signals attributable to an anomeric b-D-gluco-
pyranosyl proton (5.63 ppm, J=7.7 Hz) (Agrawal,
1992), an amide proton (10.37 ppm) and an N-acetyl
group (2.16 ppm), while the HMBC spectrum included
correlations between H-1' (5.63 ppm) and C-7 (164.2
ppm); H-9 (2.16 ppm) and C-8 (169.0 ppm); and H-2
(=H-6) (8.03 ppm) and C-7 (164.2 ppm). These data
di€erentiated the ester and amide carbonyl resonances
(164.2 and 169.0 ppm respectively) and identi®ed 4 as
N-acetyl p-aminobenzoic acid 7-O-b-D-glucopyranosyl
ester.
One- and two-dimensional NMR and electrospray
mass spectral data identi®ed the minor metabolite
recovered after incubation of o-aminobenzoic (8) as o-
amino benzoic acid 7-O-b-D-glucopyranosyl ester (6).
The 13C NMR spectrum of 6 was comprised of a
methylene, 9 methine and 3 quaternary resonances (see
1
Table 1), while the H NMR spectrum included signals
assigned to an anomeric b-D-glucopyranosyl proton
(H-1') (5.61 ppm, J=7.9 Hz) and two amine (-NH2)
protons (6.73 ppm). The HMBC spectrum of 6
included correlation between H-1' (5.61 ppm) and C-7
(166.1 ppm), H-6 (7.86 ppm) and C-2 (151.8 ppm), C-
4 (134.5 ppm) and C-7 (166.1 ppm) and the amine pro-
tons (-NH2) (6.73 ppm) and C-1 (108.1 ppm) and C-3
(116.5 ppm). The positive ion ESMS of 6, determined
in Na+ assisted matrix, showed ions of (M+H)+ and
(M+Na)+ pseudomolecular ions (m/z 300 and 322 re-
spectively), together with (2 M+H)+ and (2
M+Na)+ cluster ions (m/z 599 and 621 respectively).
The negative ion ESMS of 6, determined in a Cl
assisted matrix, showed peaks of (M±H) , (M+35Cl)
and (M+37Cl) pseudomolecular ions (m/z 298, 334
and 336 respectively), together with (2 M±H) , (2
M+35Cl) and (2 M+37Cl) cluster ions (m/z 597,
633 and 635 respectively).
The 13C NMR spectrum of the major metabolite 5,
isolated after incubation of o-aminobenzoic acid (8),
was comprised of 2 methylene, 14 methine and 3 qua-
ternary resonances (see Table 1). The 1H-NMR spec-
trum of 5 included 4 aryl proton signals, two doublet
resonances centered at 5.61 and 4.25 ppm (J=7.9 Hz
and J=7.7 Hz respectively) assigned to the anomeric
protons of two b-D-glucopyranosyl residues and a
broad two proton -NH2 signal (6.74 ppm). These data
are consistent with the formulation of 5 as a digluco-
pyranosyl analogue of 8. HMBC spectral data estab-
lished that the inner glucopyranosyl residue was
attached to the carboxyl group of the substrate and
that the pair of b-D-glucopyranosyl residues were
mutually 1,6-linked. In particular, HMBC correlations
were observed between H-1' (5.61 ppm) and C-7
(166.0 ppm), and between H-10 (4.25 ppm) and C-6'
(68.0 ppm). A notable feature of the 13C NMR spec-
trum of 5 is down®eld shift of the C-6' signal (68.0
ppm) in comparison to the resonance of the C-60 sig-
nal (60.9 ppm). This down®eld shift is consistent with
C-6' glucosylation (Agrawal, 1992). Correlations
observed in the ROESY spectrum of 5 between the H-
10 (4.25 ppm) resonance of the outer glucopyranosyl
unit and the H-6A' and the H-6B' (3.68 and 4.08 ppm
respectively) resonances of the inner glucopyranosyl
unit, and mutual correlations between the H-1', H-3'
and H-5', and the H-10, H-30 and H-50 resonances of
the inner and outer glucopyranosyl residues respect-
ively, were consistent with the identi®cation of 5 as o-
aminobenzoic acid 7-O-b-D-(b-1,6-O-D-glucopyranosyl)
glucopyranosyl ester.
3. Conclusions
To our knowledge this is the ®rst report of the bio-
transformation of 2, 7 and 8 to the b-D-glucopyranosyl
esters (1, 4, 5, or 6), by cell suspension cultures of S.
mammosum. None of these glucopyranosyl esters
appear to have been previously isolated from natural
sources. The complete assignment of the 13C and 1H
NMR resonances of the b-D-glucopyranosyl esters 1,
4, 5 and 6, given in Table 1 (13C NMR signals), or in
Section 4 (1H NMR signals) were in each case substan-
tiated by correlations observed in the two-dimensional
HSQC, HMBC, COSY and ROESY spectra of the re-
spective metabolites.
This also appears to be the ®rst report of the
isolation from cell suspension cultures of Solanum
species of a diglucopyranosyl ester, and (in the case of
p-aminobenzoic acid) the formation of N-acetyl and
N-formyl analogue. We are presently investigating the
biotransformation of other substrates by Solanum
species in order to ascertain whether or not N-acety-
lation or N-formylation is a more general biotrans-
formation phenomena in this species.
ESMS data was also consistent to the identi®cation
of 5 as a diglucopyranosyl ester. In particular, the
positive ion ESMS spectrum of 5, determined in Na+
assisted matrix, included ions attributable to (M+H)+
and (M+Na)+ pseudomolecular ions (m/z 462 and
484 respectively), together with (2 M+H)+ and (2

618
A. Syahrani et al. / Phytochemistry 51 (1999) 615±620
4. Experimental
4.3. Toxicity assessment experiments
Cells (10 g fresh weight) were inoculated into liquid
medium (50 ml) containing various concentration of 2,
7 or 8 (500, 750, 1000, 1250 and 1500 mg l 1) and con-
trol cultures (without the addition of 2, 7 or 8). After
7 days the cultures were harvested, followed by PCV
determination and microscopic observation then ®l-
tered, weighed, oven dried at 408 and powdered.
4.1. General procedures
NMR spectra were determined at 400.13 MHz (1H)
and 100.62 (13C) using an inverse 5 mm probe head
installed in a Bruker DRX 400 spectrometer, or at
300.13 MHz (1H) and 75.47 (13C) using a 5 mm QNP
probe head installed in a Bruker AC 300 spectrometer.
Gradient selection was utilized in HMBC and HSQC
experiments. Chemical shift (d ppm) are reported
relative to solvent peaks observed for DMSO-d6
(1H=2.60; 13C=39.5 ppm). Coupling constants are
reported to a precision of 20.2 Hz. 13C NMR signal
multiplicities (d, t or q: s suppressed) were determined
using the distortionless enhancement by polarization
transfer (DEPT) sequence with a 1358 detection pulse.
Two-dimensional COSY and HMBC (80 msec mixing
time) spectra were determined in absolute value mode,
while TOCSY, ROESY (250 msec spin lock time) and
HSQC spectra were determined in phase sensitive
mode. The positive and negative ion electrospray mass
spectra (ESMS) were determined using a Fissons VG
Platform II instrument. Samples were introduced into
the spectrometer using CH3CN:H2O (1:1) as solvent.
Electron impact mass spectra (EIMS) of 2 and 3 were
obtained using a Hewlett Packard (HP) HP5980B
mass selective detector (MSD) interfaced to a HP5970
GC ®tted with a 25 m  0.22 mm HP-1 capillary col-
umn. The GC oven was temperature programmed
from 1008 to 2508 at 108/min. The di€erential scanning
(DSC) thermogram of synthetic specimen of 2 was
4.4. Biotransformation of 7 and isolation of 1, 2 and 3
Incubation of p-aminobenzoic acid (7) and work-up
of the cell culture as described above, produced pow-
dered biomass (6 g) which was re¯uxed for 2 h with
MeOH. Concentration of the MeOH extract under
reduced pressure using a rotatory evaporator, a€orded
a residue (2.5 g) which was submitted to column chro-
matography (CC) on Silicagel 40 (Merck, 70±230
mesh) using EtOAc:MeOH:H2O (77:13:10) as eluent.
Puri®cation of the glucopyranosyl fraction by prepara-
tive TLC (Silicagel 60 GF 254, Merck; 0.25 mm layer)
using EtOAc:MeOH:H2O (77:13:10) as the developing
solvent and UV detection at 254 nm, yielded p-amino-
benzoic acid 7-O-b-D-glucopyranosyl ester (1) (310 mg)
(Rf 0.37) and material which was shown by one and
two-dimensional NMR and GC/MS analyses to be a
ca. 3:1 mixture of N-acetyl p-aminobenzoic acid (2)
and N-formyl p-aminobenzoic acid (3) (24 mg) (Rf
0.51).
Metabolite 1 decomposed at 1908 (DSC analyses);
positive ion ESMS (+20 V, Na+ and NH4+ assisted
matrix) m/z (% rel.int.) 300 ([M+H]+, 33), 317
([M+NH4]+, 100), 322 ([M+Na]+, 26), 616 ([2
M+NH4]+, 26), 621 ([2 M+Na]+, 14); negative ion
ESMS ( 110 V, Cl assisted matrix) m/z (% rel.int.)
298 ([M±H] , 100), 334 ([M+35Cl] , 79), 336
([M+37Cl] , 32), 597 ([2 M±H] , 61), 633 ([M+35Cl] ,
25), 635 ([M+37Cl] , 12). 1H NMR (DMSO-d6, 300
MHz) d 3.25 (1H, m, H-3'), 3.32 (1H, m, H-2'), 3.39
(2H, m, H-4' and H-5'), 3.56 (1H, m, H-6A' ), 3.73 (1H,
m, H-6B' ), 5.59 (1H, d, J=7.8 Hz, H-1'), 6.13 (2H, s,
NH2), 6.67 (2H, d, J=8.7 Hz, H-3 and H-5),
7.78 (2H, d, J=8.7 Hz, H-2 and H-6); 13C NMR: see
Table 1.
determined using
thermal analyzer.
a
Shimadzu DT30 di€erential
4.2. Cell suspension culture and biotransformation
conditions
Clone sm. of the cell suspension cultures used in
these studies were initiated from the callus cultures of
S. mammosum as previously reported (Syahrani et al.,
1997a, 1997b). The calli were cultivated in 300 ml
Erlenmeyer ¯asks containing 50 ml of modi®ed
Murashige and Skoog medium (Murashige & Skoog,
1962) supplemented with sucrose (30 g l 1), kinetin (2
mg l 1), NAA (1 mg l 1) and casein hydrolisate
(1 g l 1) on a gyrotary shaker (120 rpm) at 25 2 18
under continuous light (ca. 2000 lux). Biotrans-
formation experiments were typically performed by
inoculating cells (10 g fresh weight) into the liquid
medium (50 ml) containing 2, 7 (1000 mg l 1), or 8
(750 mg l 1) and incubated for 7 days. After 7 days
the cultures were harvested, ®ltered, weighed, oven
dried at 408 (until their water content was ca. 2%) and
powdered.
Metabolite 2 had 1H NMR (DMSO-d6, 400.13
MHz) d 2.12 (3H, s, COCH3), 7.57 (2H, d, J=8.4 Hz,
H-3 and H-5), 7.88 (2H, d, J=8.4 Hz, H-2 and H-6),
10.09 (1H, s, NH); 13C NMR (DMSO-d6, 100.62
MHz) d 24.0 (C-9), 117.5 (C-3 and C-5), 129.5 (C-2
and C-6, 134.9 (C-1), 139.8 (C-4), 168.2 (C-8), 169.7
(C-7). An authentic specimen of N-acetyl p-aminoben-
zoic acid (2) was prepared using a published method
(Vogel, 1956). The purity of the synthetic product,
which exhibited 13C NMR signals identical to observed
for the isolated specimen of 2, was veri®ed by TLC. A

A. Syahrani et al. / Phytochemistry 51 (1999) 615±620
619
sharp endothermic peak in the DSC thermogram of 2
was observed at 1478C.
solution of diazomethane, showed peaks attributable
to the presence of N-acetyl p-aminobenzoic acid
methyl ester (9), EIMS m/z (% rel.int.) 193 (M+, 29),
151 (57), 120 (100), 92 (23) and N-formyl p-aminoben-
zoic acid methyl ester (10), EIMS m/z (% rel.int.) 179
(M+, 81), 148 (68), 120 (100), 92 (32).
Metabolite 3 had 1H NMR (DMSO-d6, 400.13
MHz) d 7.58 (2H, d, J=8.4 Hz, H-3 and 5), 7.89 (2H,
d, J=8.4 Hz, H-2 and H-6), 8.34 (1H, d, J=2.0 Hz,
NH±CHO), 10.42 (1H, br s, NH); 13C NMR (DMSO-
d6, 100.62 MHz) d 117.7 (C-3 and C-5), 129.6 (C-2
and C-6), 135.6 (C-1), 138.7 (C-4), 159.2 (C-8), 169.8
(C-7).
4.5. Biotransformation of 2 and isolation of 4
GC/MS analyses of a methylated subsample of the
mixture of metabolites 2 and 3, prepared by treatment
of a CHCl3 solution of the mixture with an ethereal
Incubation of N-acetyl p-aminobenzoic acid (2) and
work-up of the cell culture as described above, pro-
duced powdered biomass (11.8 g) which was re¯uxed
Fig. 1. The structure of compounds 1±10.

620
A. Syahrani et al. / Phytochemistry 51 (1999) 615±620
for 2 h with MeOH. Concentration of the MeOH
extract under reduced pressure using a rotatory evap-
orator a€orded a residue (2.01 g) which was submitted
to column chromatography Silicagel 40 (Merck, 70±
230 mesh) using EtOAc:MeOH:H2O (77:13:10) as elu-
ent and preparative TLC (Silicagel 60 GF 254, Merck;
0.25 mm layer) using EtOAc:MeOH:H2O (77:13:10) as
the developing solvent and UV detection at 254 nm.
Removal of MeOH (used as the elution solvent for the
TLC band) yielded N-acetyl p-aminobenzoic acid 7-O-
b-D-glucopyranosyl ester (4) (117 mg) (Rf 0.25), 1H
Metabolite 6 had positive ion ESMS (+100 V, Na+
assisted matrix) m/z (% rel.int.) 300 ([M+H]+, 3), 322
([M+Na]+, 35), 599 ([2 M+H]+, 14), 621 ([2
M+Na]+, 100); negative ion ESMS ( 110 V, Cl
assisted matrix) m/z (% rel.int.) 298 ([M±H] , 73), 334
([M+35Cl] , 100), 336 ([M+37Cl] , 32), 597 ([2 M±
H] , 77), 633 ([2 M+35Cl] , 60), 635 ([2 M+37Cl] ,
1
29); H NMR (DMSO-d6, 400.13 MHz) d 3.25 (1H, m,
H-4'), 3.30±3.40 (3H, br m, H-2', H-3' and H-5'), 3.75
(1H, m, H-6A' ), 5.61 (1H, d, J=7.9 Hz, H-1'), 6.62
(1H, 0t, J=8.2 Hz, H-5), 6.73 (2H, br s, NH2), 6.86
(1H, d, J=8.4 Hz, H-3), 7.35 (1H, 0t, J=8.2 Hz, H-
4), 7.86 (1H, d, J=8.1 Hz, H-6), 3.54 (1H, m, H-6B' );
13C NMR: see Table 1. For structures of the com-
pounds used see Fig. 1.
NMR (DMSO-d6, 400.13 MHz)
d 2.16 (1H, s,
COCH3), 3.19 (1H, m, H-4'), 3.37 (3H, m, H-2', H-3'
and H-5'), 3.54 (1H, ddd, J=5.7, 5.7, 11.7 Hz, H-6'),
3.75 (1H, ddd, J=1.9, 5.7, 11.7 Hz, H-60), 4.63 (1H, t,
J=5.9 Hz, 6'-OH), 5.08 (1H, d, J=5.4 Hz, 4'-OH),
5.18 (1H, br d, J=3.1 Hz, 3'-OH), 5.41 (1H, d, J=4.7
Hz, 2'-OH), 5.63 (1H, d, J=7.7 Hz, H-1'), 7.81 (2H,
d, J=7.8 Hz, H-3 and H-5), 8.03 (2H, d, J=7.8 Hz,
H-2 and H-6), 10.37 (1H, s, NH); 13C NMR: see
Table 1.
Acknowledgements
Part of this work was supported by grants from
Ministry of Education and Cultures of the Republic of
Indonesia (TMPD S3/S2 Ð grants for AS and ER),
Ministry of Research and Technology of the Republic
Indonesia (RUT VI/1, 1998/1999) and the New
Zealand Lotteries Commission (grants for spec-
trometer purchase). The authors are very grateful to
Ms. Tiurma Susanti Panjaitan for her technical assist-
ance.
4.6. Biotransformation of 8 and isolation of 5 and 6
Incubation of o-aminobenzoic acid (8), and work-up
of the cell culture as described above, produced pow-
dered biomass (14.42 g) which was re¯uxed for 2 h
with MeOH. Concentration of the MeOH extract
under reduced pressure using a rotatory evaporator,
yielded a residue (6.01 g) which was submitted to col-
umn chromatography and preparative TLC (Silicagel
60 GF 254, Merck; 0.25 mm layer) using
EtOAc:MeOH:H2O (77:13:10) as the developing sol-
vent and UV detection at 254 nm. o-Aminobenzoic
acid 7-O-b-D-(b-1,6-O-D-glucopyranosyl)glucopyrano-
syl ester (5) (32 mg) (Rf 0.08) and o-aminobenzoic acid
7-O-b-D-glucopyranosyl ester (6) (5 mg) (Rf 0.42) were
obtained after removal of MeOH (used as the elution
solvent for the TLC band).
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