An enzyme-mimetic chemical conversion and biogenetic outline for chemical marker pterocarponoid oxygen heterocycles from Kudzu vine

Article abstract of DOI:10.1002/jhet.497

An enzymatic chemical conversion of isoflavone and chemical marker oxygen-heterocycles pterocarponoids are carried out in an analogy to major subcellular enzymatic conversions involved in the natural biogenetic pathway and microbial interaction steps for oxygen-proliferated products obtained from bioactive fractions of widely occurring Kudzu plant, Pueraria tuberosa. An outline for biogenesis of major pterocarponoid constituents from principal biogenetic precursor, deoxytuberosin, is also proposed for P tuberosa.

Full text of DOI:10.1002/jhet.497

168
An Enzyme-Mimetic Chemical Conversion and Biogenetic
Outline for Chemical Marker Pterocarponoid Oxygen
Heterocycles from Kudzu Vine
Vol 48
Riaz A. Khan*
Department of Chemistry, Manav Rachna International University (MRIU),
National Capital Region, Aravali Hills, Sector-43, Faridabad, HR 121 001, India
*E-mail: riazkhan09@hotmail.com
Received October 7, 2009
DOI 10.1002/jhet.497
Published online 6 October 2010 in Wiley Online Library (wileyonlinelibrary.com).
An enzymatic chemical conversion of isoflavone and chemical marker oxygen-heterocycles pterocar-
ponoids are carried out in an analogy to major subcellular enzymatic conversions involved in the natural
biogenetic pathway and microbial interaction steps for oxygen-proliferated products obtained from bio-
active fractions of widely occurring Kudzu plant, Pueraria tuberosa. An outline for biogenesis of major
pterocarponoid constituents from principal biogenetic precursor, deoxytuberosin, is also proposed for P
tuberosa.
J. Heterocyclic Chem., 48, 168 (2011).
INTRODUCTION
the biochemical pathways of conversions [30–32]. The
isoflavonoid biogenesis is now firmly established and
links between isoflavones and various pterocarponoid
categories have been investigated at molecular details
leading to identification of enzymes involved [33–37].
The major enzymes are membrane-bound, including
2-hydroxyisoflavanone synthase responsible for the
formation of isoflavone nucleus, pterocarpan synthase,
pterocarpan-6a-synthase (P6aH), pterocarpan-3-O-meth-
yltransferase (P3OM), prenyl transferase and prenyl
cyclase, associated with different steps of isoflavone,
and pterocarponoid biogenesis [38,39].
Isoflavonoids, an oxygen-bearing plant heterocycles,
are significant subclass of phytochemically abundant fla-
vonoid natural products and form major and vital constit-
uents of Soya and Kudzu plants derived phytomedicines
[1–5]. The plants and their parts have been found to
possess wide spectrum of biological activities including
protection against stress-induced myocardial ischemia,
nootropic activity, helpful in ameliorating conditions of
migraine and deafness, antioxidant properties, dose-de-
pendent hepatotoxicity, antimicrobial properties [6–10],
uses in estrogen replacement therapies, an emergency
food and as a food supplement [11–15]. The isoflavo-
noids play significant roles in plant–microbe interactions
at biology-environment interface [16,17] and are consid-
ered to be involved in signal transductions [18–20], con-
tributions to root-nodules formation in leguminous plants
[21–23] and their functions as plant antibiotics.
RESULTS AND DISCUSSION
During the search for postcoital anti-implantation
active phytoconstituent(s) from Pueraria tuberosa vine,
the 95% aqueous ethanolic extracts of fresh tubers
exhibiting both estrogenic and anti-estrogenic hormonal
bioactivity in mammalian test models [40], a notable
structural correlation among isolated isoflavone and
The biosynthesis of isoflavonoids has been studied in
depth including the identification of genes [24–29], iso-
lation and characterization of enzymes, and tracing of
C
V
2010 HeteroCorporation

January 2011
An Enzyme-Mimetic Chemical Conversion and Biogenetic Outline for Chemical
Marker Pterocarponoid Oxygen Heterocycles from Kudzu Vine
169
Scheme 1. Biogenetic outline.
pterocarponoids was observed. Based on oxidation states
and oxygen atom’s gradual proliferation in major isofla-
vonoid and pterocarponoid constituents in an increasing
order of molecular weight, a sequence of biogenetic
conversions is proposed (Scheme 1). A series of bio-
chemical transformations are envisioned starting from
40,7-dihydroxyisoflavone (daidzein) (1) to neobavaisofla-
vone (2), chromenoisoflavone, puerarone (3) and ptero-
carpan derivative, deoxytuberosin (4). The isolation of
these products from the plant extracts represented the
outcome of initial steps of chromeno-isoflavone biogene-
sis in the plant. The formation of pterocarpan, deoxytu-
berosin (4) as a sequel of advancing biogenesis from
daidzein (1) to neobavaisoflavone (2) formed by C-30
carbon prenylation, and C-60 carbon’s hydroxylation fol-
lowed by C-30 prenyl group cyclization to yield dime-
thylchromeno-isoflavone, puerarone (3), all established
the front-line of biogenetic sequence defining the con-
nection between isoflavone and pterocarponoid oxygen
heterocycles as well as suggesting a downstream possi-
ble route of biogenetic events. The analogous stereospe-
cific bioreduction [41,42] of intermediate isoflavone,
puerarone (3), cyclization of the resulting isoflavan-4-ol
to afford racemic pterocarpan, (6) deoxytuberosin (4),
and transformation of this principal pterocarponoid
biogenetic precursor, (6) deoxytuberosin (4) to other
chromeno-pterocarpanoids by incorporating molecular
oxygen into the racemic pterocarpan (4) to predictably
produce pterocarpan-6a-ol(s) (5) and (6), namely isotu-
berosin [(ꢀ) tuberosin (5)] and tuberosin [(þ) tuberosin
(6)] is in line with other studies reported on leguminous
plants involving different and nearly similar structural
templates of pterocarponoid biosynthetic patterns
including initial proposals [41–44] for P. tuberosa.
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R. A. Khan
Vol 48
Furthermore, in the later events, a possible monooxy-
genase mechanism involving pterocarpan-6a-ol, tubero-
sin (6) to produce pterocarpanone, 1a-hydroxytubero-
sone (7) again substantiated the biogenetic proposal,
Scheme 1. The dehydrogenation of deoxytuberosin (4)
to yield anhydrotuberosin (8), which by late biogenetic
O-methylation produces 3-O-methylanhydrotuberosin
(8a) and the biochemical allylic oxidation of the C-6-
methylene function in 3-O-methylanhydrotuberosin (8a)
resulting in the production of pterocarpone/coumestan
nucleus, tuberostan (9), provided the pterocarpene and
coumestan class of pterocarponoid compounds respec-
tively in the progressing biogenetic line. The isolation
of newly found isotuberosin (5) from this plant and
tuberosin (6), both from the bioactive chloroform
extracts of Pueraria tuberosa was conclusive of the
involvement of stereospecific pterocarpan precursor
deoxytuberosin (4), isolated here as a racemic mixture,
to produce these pterocarpan-6a-ol(s) (5) and (6), and
thus, unequivocally establishing the involvement of
stereo-specific reduction of isoflavone transformation in
this plant also. The pterocarpan, deoxytuberosin (4), and
pterocarpan-6a-ol isotuberosin (5) were isolated in trace
amounts from the plant when compared with pterocar-
pan-6a-ol, tuberosin (6), 1a-hydroxytuberosone (7),
anhydrotuberosin (8), and 3-O-methylanhydrotuberosin
(8a). The insignificant concentrations of product (4)
strongly indicated the possibility of its involvement as
an intermediate template-pool of molecule for advancing
the biogenesis in producing new pterocarponoid com-
pounds. The probable participation of products (4) and
(5) as in situ antimicrobial agent and (6) being tested
and a known antimicrobial agent against certain plant-
pathogens, indicated the possibilities of stimulus-respon-
sive plant biogenesis of secondary metabolites. The
highly oxygenated pterocarpanone subclass of pterocar-
ponoids in comparison with pterocarpan-6a-ol (constitu-
ent oxygen ratio, 27.09:23.64, respectively), also classi-
fied as ‘‘stress metabolites,’’ are generally considered to
be biogenetically derived from pterocarpan-6a-ol by
oxygen proliferation owing to its participation in the
detoxification process in response to external stimuli
(e.g., microbes, wounds, etc.) in the plants’ environment
[45]. The generation of 1a-hydroxytuberosone (7) from
tuberosin (6) indicated likewise but, the generation of
these classes of pterocarponoids is also encountered in
other leguminous plants, the possibility of them being
generated on a genetically planned biogenetic lines as
part of leguminous plant defense seems more plausible
as their occurrence can not be considered specific to
some leguminous plants. It is also suggested that deoxy-
tuberosin (4) is the principal phytoalexin (plant antibi-
otic) of P. tuberosa and immediate biogenetic precursor
of pterocarpan-6a-ol (5) and (6), wherein both probably
are a product of milder external stimuli and hereby sug-
gesting a response-related biogenesis. The product (6)—
a tested and known antimicrobial active constituent,
is produced as a milder-active phytoalexin, whereas
the pterocarpanone, 1a-hydroxytuberosone (7) is the
final product of the stimulus generated biochemical
response’s detoxification process common to a variety
of leguminous plants. A study showing weak antifungal
activity of tuberosin (6) and no antifungal activity in 1a-
hydroxytuberosone (7) also indicated likewise for its
plant defense roles and the bioecological outcome.
Again, the term ‘‘stress metabolite’’ is also applicable to
pterocarpone or coumestan oxygen heterocylces, tubero-
stan (9), which is a product of allylic oxidation of 3-O-
methylanhydrotuberosin (8a) in response to certain stim-
ulus [46,47], whereas pterocarpene, anhydrotuberosin
(8), and 3-O-methylanhydrotuberosin (8a) are envi-
sioned to be produced from pterocarpan deoxytuberosin
(4) and/or 3-O-methyldeoxytuberosin (4a), Scheme 1.
The anhydrotuberosin (8) and 3-O-methylanhydrotubero-
sin (8a) represented the pterocarpene class, whereas
tuberostan (9) and 1a-hydroxytuberosone (7) were iden-
tified as pterocarpone and pterocarpanone subtypes of
pterocarponoid compounds, respectively, from this plant
[48–51].
However, in the more complex picture of isoflavonoid
biogenesis and detoxification process, the occurrence of
quantitatively minor isoflavone and pterocarponoid com-
pounds with varying substitution patterns, incorporating
hydroxyl, methoxyl, prenyl (C3H9), and analogs, oxida-
tion(s) of substituent groups, and other chemical entities
as substitutions, can be considered an outcome of aber-
rant biogenesis which seems to be running on a non-pre-
determined biogenetic plan and thereby producing new
structural variants of the known classes and/or individ-
ual compounds as a specific result of response-mediated
biogenetic action on demand because of the plant and
its surroundings’ interplay of either symbiotic nature or
of hostile intentions. The presence of isoflavonoid glyco-
sides with variations in glycone(s) positions, differences
in glycone’s esterified or etherified residues’ nature in
terms of alkyl structural types/chain length incorporation
of other simple aromatics (e.g., substituted phenyls,
cinnamic acids, etc.) and nonaromatic entities (e.g., ter-
penic, aliphatic residues) into the glycone part or attach-
ment/fusion in aglycone moiety itself may also be an in-
dication of response-mediated natural deviation from the
planned biogenetic program. An array of plant products
with biogenetically plausible and/or structurally varying
isoflavonoid and pterocarponoids and their glycoside(s)
for use as plant defencins, antifeedants, plant-antibiotics
(phytoalexins), anti-inducers, and toxins are part of sur-
vival mechanism against variety of microbial pathogens,
physicochemical and plant physiological stress factors in
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An Enzyme-Mimetic Chemical Conversion and Biogenetic Outline for Chemical
Marker Pterocarponoid Oxygen Heterocycles from Kudzu Vine
171
Scheme 2. Enzyme-mimetic chemical conversions.
and around the plant ecosystem, wherein the demarca-
tion between routine biogenesis and stimuli-responsive
generation of natural products is hard to distinguished.
Moreover, the biogenesis of an entirely different chemi-
cal class of compound produced from the biogenetic
precursor-pool of common chemical entities but beyond
the established biogenetic plan of producing the chemi-
cal markers and other species-specific compounds
including compounds involved or produced as a function
of plants’ normal physiological conditions dictated by
its genetic make-up, is probably because of earliest
response towards the external or consequent internal
stimuli passivation based upon its severity for the plant
survival.
vone*, and an unidentified dimeric isoflavonoid product
(Mþ.678) found during this study (*new to this plant) as
well as the reported presence of 40,6"di-O-acetylpuerarin
[56] from P. tuberosa are noteworthy for their possible
roles in plant microbe interplay and their further bioge-
netic interests with regard to the enzymatic participation
in biochemical duress conditions of external stimuli.
Herein, an attempt is made to find the common chemical
ground for the major and characteristic chemical marker
pterocarponoid constituents of P. tuberosa and mimic
the different product-based enzymatic out-comes of the
biogenesis to understand the analogy between the chem-
ical and enzymatic transformations and their feasibilities
at chemical levels.
Thus, the isolation of puerarostan [52], puetuberosa-
nol [53,54], dihydrodaidzein, p-hydroxybenzoic acid*,
3-methoxy-9-hydroxypterocarpene*, 3-O-methyldeoxytu-
berosin*, maltol*, 40,7, 8-trihydroxyisoflavone* [55],
puerarin, daidzin, 40-hydroxy–7-O-rhamnosyl-isofla-
The correlation of major isoflavones and pterocarpo-
noid chemical marker compounds representing the
advancing biogenetic transformation sequence among
the 40,7-dioxygenated isoflavone and 3,9-dioxygenated-
2,2-dimethylchromeno-pterocarponoid
classes
are
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172
R. A. Khan
Vol 48
Enzyme-mimetic chemical conversion of isoflavone and
pterocarponoid constituents. The fresh tubers collected from
Shahdol forests, MP, India were extracted with 95% aqueous
ethanol under 40ꢁC overnight, evaporated to dryness under
vacuum as a red-brown gelly and partitioned between aqueous
and hexane, chloroform, n-BuOH fractions and residual water
portion. A part of the chloroform soluble fraction was repeat-
edly column chromatographed over silica-gel (23–400 mesh)
in increasing polarities of hexane and EtOAc, neat EtOAc, and
EtOAc-MeOH. All the isolated new and known products were
well-characterized by MS, PMR, elemental analysis, specific
rotations, IR and UV spectro-analytical methods, and circular
dichroism measurements wherever needed and with thorough
comparisons of m.p., mixed m.p., co-IR, co-UV determinations
with the known reference standards of single spot TLC purity
samples (elementally pure) obtained commercially or isolated
and characterized during the course of this study (Table 1).
Puerarone (3) to Deoxytuberosin (4). To a solution of
puerarone (3, 25 mg) in dry methanol (10 mL), sodium-borohy-
dride (50 mg) was added and stirring continued, after 30 min,
solvent was removed to furnish a residue, which was diluted
with water, acidified with 5% aq. HCl, and extracted with chlo-
roform (2 ꢂ 25 mL). The combined organic layers were dried
over anhydrous sodium sulphate and conc. to give isoflavan-4-
ol as oil. UV (MeOH): 230, 280, 290, 320 nm. MS (m/z): 340
(Mþ), 187 and 138. To the isoflavan-4-ol dissolved in dichloro-
methane (20 mL) was added glacial acetic acid (1.0 mL) and
RM refluxed at water bath for 1 h. Solvent evaporated under
vacuum to yield a solid, which was PLC purified to give deoxy-
tuberosin (4), identical in all respects to the natural sample,
amorphous solid, m p., 205ꢁC, Co-TLC, Co-UV, and Co-IR.,
UV(MeOH): 220, 280, 330, and 310 nm. UV (MeOH þ
NaOH): 220, 282, 315, and 347 nm. IR (KBr): 3400 (br), 1590,
1260, 1045, 890 cmꢀ1. Elem. Anal., calculated for C20H18O4: C
74.52, H 5.63, O 19.85, observed: C 74.54, H 5.66, O 19.89
chemically mimicked (Scheme 2) by formation of deox-
ytuberosin (4) from puerarone (3) [52,57] through C-3,4
a,b-unsaturated carbonyl’s reduction and subsequent cy-
clization. The conversion of pterocarpan to pterocarpan-
6a-ol is mimicked on template molecule, 3,9-dimethox-
ypterocarpan (11) after repeated attempts to produce
tuberosin (6) or isotuberosin (5) [58] from deoxytubero-
sin (4) failed. The oxidative conversion of 3,9-dimethox-
ypterocarpan (11) to 3, 9-dimethoxypterocarpan-6a-ol
(12) is carried out using lead tetra acetate-aqueous acetic
acid. The conversion of tuberosin (6) to yield 1a-
hydroxytuberosone (7) is achieved from sodium period-
ate after many attempts with Fremy’salt, thallium(III)
triperchlorate, and thallium (III) triflouro acetate were
unsuccessful to produce the desired compound, (7). The
further transformations to 3-O-methyldeoxytuberosin
(4a) from deoxytuberosin (4), 3-O-methylanhydrotubero-
sin (8a) from anhydrotuberosin (8) and vice versa (8a to
8) is mimicked using methyl iodide for the methylation
step in an analogy to the biogenesis of 3-O-methylptero-
carpan and 3-O-methylpterocarpene from pterocarpan
and pterocarpene, whereas boron tribromide [59] (Haz-
ard!) was used for the demethylation step to produce
back the pterocarpene (8) from (8a). The pterocarpone,
tuberostan (9) is obtained by DDQ oxidation of 3-O-
methylanhydrotuberosin (8a)—a known method [43].
However, attempts to produce pterocarpene, anhydrotu-
berosin (8) from pterocarpan, deoxytuberosin (4) by
dehydrogenation and production of 40,7,8 trihydroxyiso-
flavone (10) from 40,7-dihydroxyisoflavone (daidzein)
(1) by potassium persulfate oxidation in acetone-water
did not yield the desired products.
Deoxytuberosin
(4)
to
3-O-methyl-deoxytuberosin
(4a). Deoxytuberosin (4, 20 mg) on methylation in refluxing
acetone (20 mL) with methyl iodide (0.5 mL) and anhydrous
K2CO3 (30 mg) gave an amorphous solid on work up, which
was identified as 3-O-methyl-deoxytuberosin (4a, 12 mg) on
comparisons with the natural sample., m p., 187ꢁC, Co-TLC,
Co-UV, and Co-IR, UV (MeOH): 305, 335, and 322 nm. UV
(MeOH þ NaOH): 245, 282, 345, and 367 nm. IR (KBr):
1596, 1240, 1065, 790 cmꢀ1. Elem. Anal., calculated for
C21H20O4: C 74.98, H 5.99, O 19.03, observed: C 75.02, H
6.01, O 19.06.
This study provided the chemical correlation among
the major isoflavone and kudzu vine’s characteristic
chemical marker pterocarponoid class of compounds
with a view on biogenetic interrelationship and helped
in establishing the chemical analogy for the enzyme-
mimetic conversions of these compounds and their
natural encounter in the plant as part of bioecology
of plant microbe interactions and detoxification
processes.
Tuberosin (6) to 1a-hydroxytuberosone (7). To tuberosin
(6, 30 mg), excess sodium periodate (20 mg) in water (50 mL)
was added and stirred for 10 h. The light-brown mixture
evaporated to 1/3 volume, extracted with diethyl ether (2 ꢂ 25
mL), dried over anhydrous sodium sulfate and concentrated to
an oily mass, which was purified by PLC to give 1a-hydroxy-
tuberosone, identical to natural sample (7, 22 mg), m. p.
133ꢁC, (a)D þ117ꢁ (c ¼ 0.2, MeOH). Co-TLC, Co-UV, Co-
IR, mixed m.p., UV (MeOH): 222, 280, 308, and 325 nm. IR
(Neat): 3450, 1665, 1485, 1020, and 760 cmꢀ1. Elem. Anal.,
calculated for: C20H18O6: C 67.79, H 5.12, O 27.09, observed.
C 67.82, H 5.14, O 27.11.
EXPERIMENTAL
General. The melting points are uncorrected and expressed
in degree Celsius. The UV spectra were recorded on Hitachi-
320 spectrophotometer and IR spectra on Perkin-Elmer 157
instrument as KBr pellets, unless otherwise stated. The PMR
and mass spectra were recorded on Brucker WM 400-MHz
spectrometer and Jeol JMS D-300 instrument in EI mode at 70
eV, respectively. The precoated silica gel TLC plates (Merck,
Darmstadt) were used and visualized by short UV wavelength
and/or on spraying with 2N sulphuric acid solution of ceric
sulfate and subsequent heating.
1a-Hydroxytuberosone (7) to tuberosin (6). A mixture of
1a-hydroxytuberosone (7, 10 mg), Zn-dust (20 mg) and glacial
acetic acid (4.0 mL) was vigorously stirred at RT for 2 h and
Journal of Heterocyclic Chemistry
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Table 1
Spectro-analytical data of major compounds.
NMR data
Molecular formula, MW
(Mþ) and mass peaks (m/z)
1H NMR:Solvent-dx,TMS as internal reference, chemical shifts
interchangeable and expressed in d values, coupling constants in Hz
Sr. No.
1
Compound
Daidzein
C15H10O4, 254 (Mþ), 137,
Acetone-d6 þ DMSO-d6: 6.65 - 6.96 (m, 3H, C30H, C50H, C6H),
7.04 -7.30 (m, 2H, C20H, C8H), 7.88 (d, 1H, J ¼ 8.5 Hz, C5H),
8.76 (s, 1H, C2H).
118
2
3
Neobavaisoflavone
Puerarone
C20H18O5, 338 (Mþ), 323,
253, 161
Acetone-d6: 6.56 (m, 3H, C20H, C60H, C6H), 6.99-7.15 (m, 2H, C50H,
C8H), 7.79 (d, 1H, J ¼ 8.5 Hz, C5H), 8.89 (s, 1H, C2H).
Acetone-d6: 1.40 (br s, 6H, 2 ꢂ CH3), 5.62 (d, 1H, J ¼ 10.0 Hz,
C13H), 6.35 (d, 1H, J ¼ 10.0 Hz, C12H), 6.37 (s, 1H, C7H), 7.02
(d, 1H, J ¼ 2.0 Hz, C8H), 7.04 (s,1H, C10H), 7.20 (dd, 1H, J ¼ 8.5,
2.0 Hz, C6H), 8.143 (d, 1H, J ¼ 8.5 Hz, C5H), 8.34 (s, 1H, C2H).
Acetone-d6: 1.24 (br s, 6H, 2 ꢂ CH3, isoprenyl), 3.22 (m, 1H, C6H),
4.02 (m, 2H, C6aH ꢂ 2), 5.16 (m, 1H, C11aH), 5.64 (d, 1H, J ¼ 10.0
Hz, C13H), 6.37 (d, 1H, J ¼ 10.0 Hz, C12H), 6.67 (dd, 1H, J ¼ 8.5,
2.5 Hz, C2H), 7.00 (s, 1H, C10H), 7.22 (d, 1H, J ¼ 2.5 Hz, C4H),
7.38 (s, 1H, C7H), 7.42 (d, 1H, J ¼ 8.5 Hz, C1H).
C20H16O5, 336 (Mþ), 321
(Mþ-CH3), 170, 134
4
5
6
7
Deoxytuberosin
C20H18O4, 322 (Mþ), 307,
173, 149, 136
1a-Hydroxytuberosone C20H18O6, 354 (Mþ), 339,
Acetone-d6: 1.37 (s, 6H, 2 ꢂ CH3), 4.40 (d, 1H, C6a-H, J ¼ 11 Hz),
4.80 (s, 1H, C11 aH), 5.12 (d, 1H, C6bH, J ¼ 11 Hz), 5.30 (d, 1H,
C4H, J ¼ 2.0 Hz), 5.60 (d, 1H, C13, J ¼ 10 Hz), 6.02 (dd, 1H, C2H,
J ¼ 10.0, 2.0 Hz), 6.10 (s, 1H, C10H), 6.42 (d, 1H, C12H, J ¼ 10
Hz), 6.90 (d, 1H, C1H, J ¼ 9 Hz), 7.10 (s, 1H, C7H).
CDCl3: 1.37 (s, 6H, 2 ꢂ CH3), 4.00 (d, 1H, J ¼ 11.0 Hz, C-6H), 4.02
(d, 1H, J ¼ 11.0 Hz, C60H ), 5.20 (s, 1H, C11H), 5.60 (d, 1H, J ¼
10.0 Hz, C12H), 6.25 (s, 1H, C7H ), 6.26 (d,1H, J ¼ 2.0 Hz, C4H),
6.40 (d, 1H, J ¼ 10.0 Hz, C13 H), 6.50 (dd, 1H, J ¼ 8.5, 2.0 Hz,
C2H), 7.02 (s, 1H, C10 H), 7.25 (d, 1H, J ¼ 8.5 Hz, C1H).
CDCl3: 1.46 (s, 6H, 2 ꢂ CH3), 3.81 (s, 3H, OCH3), 5.52 (s, 2H, C-6H),
5.63 (d, 1H, C-13H, J ¼ 10 Hz), 6.40 (d, 1H, C12H, J ¼ 10.0 Hz),
6.50 (dd, 1H, C2H, J ¼ 8.5, 2.5 Hz), 6.52 (d, 1H, C4H, J ¼ 2 Hz),
6.70 (s, 1H, C10H), 7.00 (d, 1H, C1H, J ¼ 8.5 Hz), 7.14 (s, 1H,
C7H).
320, 305, 201, 163
Tuberosin
C20H18O5, 338 (Mþ),
320,305,149.
3-O-Methylanhydro-
tuberosin
C21H18O4, 334 (Mþ), 319
(Mþ-CH3, 100 %), 295
8
9
Anhydrotuberosin
Tuberostan
C20H16O4 320 (Mþ), 305,
CDCl3:1.43 (s, 6H, 2 ꢂ CH3), 5.53 (s, 2H, C6H), 5.70 (d, 1H, C13H,
J ¼ 10 Hz), 6.45 (d, 1H, C12H, J ¼ 10 Hz), 6.50 (d, 1H, C4H, J ¼ 2
Hz), 6.54 (dd,1H, C2H, J ¼ 8.5, 2.5 Hz), 6.82 (s, 1H, C10H), 7.10
(s, 1H, C 7H), 7.30 (d, 1H, C1H, J ¼ 8.5 Hz).
295, 136
C21H16O5, 348 (Mþ), 347,
333(100%), 318, 262, 202,
167, 145.
CDCl3:1.43 (brs, 6H, 2 ꢂ CH3), 3.81 (s, 3H, OCH3), 5.75 (d, 1H, 10
Hz, C13H), 6.50 (d, 1H, 10 Hz, C12H), 7.15 (m, 2H, C3H, C5H),
7.30 (s, 1H, C10 H), 7.95 (dd, 1H, J ¼ 8.5, 2.5 Hz, C2H), 8.02 (s,
1H, C7H).
10
11
8-Hydroxy daidzein*
C15H10O5, 270 (Mþ), 152,
118
Acetone-d6: 6.70-6.90 (m, 4H, ring-B, C30H, C40H, C50H, C60H), 7.35
(d, 1H, C6H, J ¼ 8.5 Hz), 7.99 (d, 1H, C8H, J ¼ 8.5 Hz), 8.00
(s,1H, C2H).
3,9-Dimethoxy-
pterocarpan
C17H16O4, 300 (Mþ), 285,
284, 256, 241, 224, 186,
163, 148
CDCl3: 3.58 (s, 3H, OCH3), 3.22 (m, 1H, C 6a), 3.99 (m, 2H, C 6H),
5.11 (m, 1H, C 11aH), 6.64 -6.77 (m, 2H, C2H, C8H), 6.86 (s, 1H,
C10H), 7.00 (d, 1H, J ¼ 2.0 Hz, C4H), 7.22 (s, 1H, C7H), 7. 26
(d, 1H, J ¼ 8.5 Hz, C1H).
12
13
6a-Hydroxy-3,
9-dimethoxy-
pterocarpan
C17H16O5, 300 (Mþ), 285,
284, 256, 241,
224,186,163, 148.
CDCl3: 3.77 (s, 6H, 2 ꢂ OCH3), 4.55 (brd, 2H, C6H, J ¼ 11.5, Hz),
5.72 (s, 1H, C11aH), 6.65 (m,2H, C8H, C10 H), 7.01 (m, 2H, C3H,
C5H), 7.55 (dd, 1H, C2H, J ¼ 8.5, 2.0 Hz), 7.78 (d, 1H, C7H, J ¼
8.6 Hz).
Isotuberosin*
C20H18O6, 338(Mþ), 323,
CDCl3: 1.37 (s, 6H, 2 ꢂ CH3), 4.02 (d, 1H, J ¼ 11.0 Hz, C-6H), 4.05
(d, 1H, J ¼ 11.0 Hz, C60H ), 5.25 (s, 1H, C11H ), 5.54 (d, 1H, J ¼
10.0 Hz, C12H), 6.20 (s, 1H, C7H ), 6.30 (d, 1H, J ¼ 2.0 Hz, C4H),
6.38 (d, 1H, J ¼ 10.0 Hz, C13 H), 6.55 ( dd, 1H, J ¼ 8.5, 2.0 Hz,
C2H), 7.05 (s, 1H, C10 H), 7.28 (d, 1H, J ¼ 8.5 Hz, C1H)., [a]D
þ11.0 (c ¼1, acetone), Circular Dichroism, MeOH: þ 262, 284, 290,
340, Oil.
305, 295, 279, 256, 149.
14
3-O-Methyl-
deoxytuberosin*
C21H20O4, 336(Mþ), 321
(Mþ -CH3, 100 %),
307, 149.
CDCl3: 1.37 (br s, 6H, 2 ꢂ CH3, isoprenyl), 3.25 (m, 1H, C6H), 3.55
(s, 3H, Aryl-OCH3), 4.00 (m, 2H, C6a H ꢂ 2), 5.20 (m, 1H, C11aH),
5.70 (d, 1H, J ¼ 10.0 Hz, C13H), 6.42 (d, 1H, J ¼ 10.0 Hz, C12H),
6.70 (dd, 1H, J ¼ 8.5, 2.5 Hz, C2H), 7.00 (s, 1H, C10H), 7.20
(d, 1H, J ¼ 2.5 Hz, C4H), 7.41 (s, 1H, C7H), 7.42 (d, 1H,
J ¼ 8.5 Hz, C1H).
* New to plant.

174
R. A. Khan
Vol 48
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water (15 mL) was added slowly followed by extraction with
chloroform (2 ꢂ 15 mL). The combined extracts were dried
over anhydrous sodium sulfate and solvent removed under vac-
uum to afford tuberosin (6 mg). m.p. 211ꢁC, UV (MeOH): 220,
280, 312, 318, and 325 nm., IR: 3250, 1610, 1560, 1380, 1160,
[4] Wang, H. J.; Murphy, P. A. J Agric Food Chem 1994, 42,
1666.
1040, 800, 760 cmꢀ1. Elem. Anal., calculated for C20H18O5:
70.99, H 5.36, O 23.64, observed. C 71.00, H 5.36, O 23.65.
C
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Anhydrotuberosin (8) to 3-O-methylanhydrotuberosin (8a). To
a solution of anhydrotuberosin (8, 20.0 mg) in dry acetone (25
mL) was added methyl iodide (1.0 mL), potassium carbonate (25
mg), and reaction mixture refluxed for 1 h, cooled, solvent
evaporated to furnish a residue, which was dissolved in diethyl
(20 mL), washed with water, organic layers dried over anhydrous
sodium sulfate, and evaporated to give the product (8a, 12 mg),
identical in all respect to natural sample. m.p. 165ꢁC, TLC, Co-T
LC UV, FT-IR, Co-UV, Co-IR, mixed m.p., UV (MeOH, max):
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230, 245, 332, 360 nm. Elem. Anal., calculated for: C21H18O4:
75.43, H 5.43, O 19.14., Observed. C 75.45, H 5.44, O 19.16.
C
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3-O-Methylanhydrotuberosin (8a) to anhydrotuberosin (8). To
a stirred solution of cooled (dry ice-acetone, ꢀ78ꢁC) 3-O-meth-
ylanhydrotuberosin (8a, 20 mg) in dry dichloromethane (15
mL) was added boron tri bromide in excess (2 mL) via syringe
and stirring continued for 30 min. The reaction mixture was
allowed to warm to RT and the concentrated residue was
washed with water, CHCl3 extracted, dried over anhydrous so-
dium sulfate and concentrated under vacuum to give the product
(8, 12 mg), identical to natural sample in all respects. m.p.
187ꢁC, Co-TLC, Co-UV, Co-IR, mixed m.p., UV (MeOH):
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C
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