C-Glucosylflavonoid biosynthesis from 2-hydroxynaringenin by Desmodium uncinatum (Jacq.) (Fabaceae)

Article abstract of DOI:10.1016/j.tetlet.2009.07.118

[2′,3′,5′,6′-²H₄]-2-Hydroxynaringenin is synthesised and incubated with commercially available UDP-glucose and the crude protein extract from Desmoduim uncinatum leaves. The organic extract produces isotopically labelled [2′,3′,5′,6′-²H₄]-vitexin and [2′,3′,5′,6′-²H₄]-isovitexin. Repeating the experiment with denatured protein or replacing the 2-hydroxynaringenin with [2′,3′,5′,6′-²H₄]-apigenin or [2′,3′,5′,6′-²H₄]-naringenin results in no observable incorporation. 2-Hydroxynaringenin is therefore the substrate for C-glucosylflavonoid biosynthesis in D. uncinatum.

Full text of DOI:10.1016/j.tetlet.2009.07.118

Tetrahedron Letters 50 (2009) 5656–5659
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
C-Glucosylflavonoid biosynthesis from 2-hydroxynaringenin
by Desmodium uncinatum (Jacq.) (Fabaceae)
*
Mary L. Hamilton, John C. Caulfield, John A. Pickett, Antony M. Hooper
Biological Chemistry Department, Centre for Sustainable Pest and Disease Management, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
a r t i c l e i n f o
a b s t r a c t
[20,30,50,60-2H4]-2-Hydroxynaringenin is synthesised and incubated with commercially available UDP-glu-
cose and the crude protein extract from Desmoduim uncinatum leaves. The organic extract produces
isotopically labelled [20,30,50,60-2H4]-vitexin and [20,30,50,60-2H4]-isovitexin. Repeating the experiment with
denatured protein or replacing the 2-hydroxynaringenin with [20,30,50,60-2H4]-apigenin or [20,30,50,60-2H4]-
naringenin results in no observable incorporation. 2-Hydroxynaringenin is therefore the substrate for C-
glucosylflavonoid biosynthesis in D. uncinatum.
Article history:
Received 26 June 2009
Revised 15 July 2009
Accepted 20 July 2009
Available online 24 July 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Desmodium uncinatum
C-Glycosyltransferase
Biosynthesis
C-Glycosylflavonoid
2-Hydroxynaringenin
Vitexin
Isovitexin
Deuterium labelling
Desmodium uncinatum (Jacq.) is used as an intercrop in subsis-
tence farming of maize (Zea mays) in East Africa where it has dem-
onstrated effectiveness in the suppression of parasitism by the
parasitic plant Striga hermonthica (Del.) Benth., a weed that can
devastate crops in the region.1–4 The effect was shown to be allelo-
pathic5 and during investigations into the mechanism, we have
discovered C-glycosylflavonoids, in particular the di-C-glycosylfl-
and more recently in wheat, Triticum aestivum L. (Poaceae) and rice,
Oryza sativa (Poaceae).12
To study C-glycosylflavonoid biosynthesis in D. uncinatum we
completed a total synthesis of 2-hydroxynaringenin (1) using a
rearrangement as the key to creating the carbon skeleton (Scheme
1). 6-Hydroxy-2,4-dibenzyloxyacetophenone (2) was prepared by
benzylation of triacetoxyphloroglucinol13 and acylation followed
by monodeprotection. Esterification with 4-benzyloxybenzoic acid
gave 3, a substrate that yields 4 after the Baker–Venkataraman
rearrangement. Deprotection produces 2-hydroxynaringenin (1)
which could be further converted into apigenin (5) by dehydration
in mild acid and this was hydrogenated to naringenin (6), with a
side product being dihydronaringenin chalcone. NMR analysis of
1 was performed at ꢀ60 °C in acetone at which temperature the
interconverting ring-closed and open-chain tautomers were dis-
tinct on the NMR timescale.14 The reaction sequence was repeated
using [2,3,5,6-2H4]-4-benzyloxybenzoic acid prepared from
[2,3,4,5,6-2H5]-phenol (Scheme 2)15 to yield the labelled materials
[20,30,50,60-2H4]-1, [20,30,50,60-2H4]-5 and [20,30,50,60-2H4]-6.
avonoid, 6-C-a-L-arabinopyranosyl-8-C-b-D-glucopyranosylapige-
nin (isoschaftoside), in the root extract and root exudates of D.
uncinatum that affect the early stages of Striga development.6
Elucidating the biosynthetic pathway for this class of compound
and characterising the enzymes that control C-glycosylflavonoid
biosynthesis provides the potential for transferring the mechanism
for Striga protection from this cattle forage legume into an edible
crop legume,6 and C-glycosyltransferase (CGT) activity is a key role.
Previous biosynthetic studies have shown incorporation of radiola-
belled naringenin and p-coumaric acid but not apigenin into C-
glucosylflavones by Swertia japonica (Gentianaceae)7 and Spirodela
polyrhiza (Lemnaceae).8,9 This work used whole plants and did not
preclude the possibility of a 2-hydroxyflavanone as the glucosyla-
tion substrate. Later work showed incorporation of 2-hydroxyflav-
anones into C-glucosylflavones by a protein preparation from
buckwheat cotyledons, Fagopyrum esculentum (Polygonaceae)10,11
Incubation of [20,30,50,60-2H4]-1 with UDP-glucose and a pro-
tein extract isolated from D. uncinatum leaves was per-
formed.16,17 The organic residue was purified by HPLC to afford
vitexin (8-C-b-
D-glucopyranosylapigenin) and isovitexin (6-C-b-
D-glucopyranosylapigenin) which were produced during the as-
say. Electrospray mass spectrometry showed that during the
* Corresponding author. Tel.: +44 1582 763133; fax: +44 1582 762595.
E-mail address: tony.hooper@bbsrc.ac.uk (A.M. Hooper).
incubation with leaf protein [20,30,50,60-2H4]-1 was converted into
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.07.118

M. L. Hamilton et al. / Tetrahedron Letters 50 (2009) 5656–5659
5657
BnO
OBn
HO
OH
BnO
OBn
O
BnO
OBn
(i)-(ii)
(iii)-(iv)
(v)
OBn
O
O
O
OH
OH
OBn
2
3
OH
OH
OH
HO
OH
O
HO
O
BnO
OBn
OBn
(vii)
HO
O
(ix)
(vi)
(viii)
OH
O
OH
O
OH
OH
O
OH O
4
1
5
6
Scheme 1. Synthesis of 2-hydroxynaringenin (1). Reagents and conditions: (i) Ac2O, pyridine; (ii) NaH, BnCl, H2O, quant. 2 steps; (iii) AcOH, TFAA, 0 °C, 87%; (iv) TiCl4, 76%;
(v) water-soluble DCC, 4-benzyloxybenzoic acid, DMAP; (vi) KOH, pyridine, 75 °C, 67%; (vii) H2, 10% Pd/C, 65%; (viii) MeOH, few drops 3 N HCl, quant.; (ix) H2, 10% Pd/C, 25%.
OBn
OH
OBn
[2',3',5',6'-2H4]-1
[2',3',5',6'-2H4]-5
[2',3',5',6'-2H4]-6
D
D
D
D
D
D
D
D
D
D
D
D
(i)-(ii)
(iii)
Scheme 1
D
Br
O
OH
Scheme 2. Synthesis of [2,3,5,6-2H4]-4-benzyloxybenzoic acid. Reagents and conditions: (i) Br2, dioxane; (ii) K2CO3, BnBr, 63% over 2 steps; (iii) Mg, CO2, 41%.
both [20,30,50,60-2H4]-vitexin and the regioisomer [20,30,50,60-2H4]-
isovitexin (Fig. 1). A small quantity of unlabelled vitexin and iso-
vitexin was present in the labelling experiments and protein
controls as they are water soluble and co-extracted with the leaf
proteins. In further experiments [20,30,50,60-2H4]-5 and
[20,30,50,60-2H4]-6 were not converted into [20,30,50,60-2H4]-vitexin
(Fig. 2) and so are not C-glucosylation substrates. There was
no conversion of [20,30,50,60-2H4]-1 into vitexin or isovitexin in
the controls without the addition of UDP-glucose, nor in experi-
ments containing protein denatured by boiling water, thereby
demonstrating glucosylation to be enzyme-mediated.
mechanism of Streptomyces fradiae C-glycosyltransferase, UrdGT2,
in which the Asp137 residue removes a phenolic proton, stabilising
the substrate and increasing the nucleophilicity.18 [20,30,50,60-2H4]-
1 is a symmetric substrate when in the open-chain form and also
after glucosylation, so the production of both C-glucosylflavones,
vitexin and isovitexin, may result either from chemical dehydra-
tion of the symmetric glucosylated intermediate or by dehydration
controlled by the C-glycosyltransferase (CGT) or a CGT-indepen-
dent dehydratase.
The substrate for C-glucosylation has been demonstrated to be
1 in Poacaea12 and Polygonaceae,10,11 and other work in the Lemn-
aceae8,9 and Gentianaceae7 is consistent. We have completed a
high yielding total synthesis of 1, which can be adapted to produce
substrate analogues, and used stable isotopic labelling of 1 to dem-
onstrate that it is the C-glucosylation substrate for the biosynthesis
of C-glucosylflavones vitexin and isovitexin in D. uncinatum (Faba-
[20,30,50,60-2H4]-1 exists as a mixture of tautomers and either the
open-chain or the ring-closed form may be C-glucosylated. The
nucleophilicity of the aromatic ring to facilitate C-glucosylation
could be increased by a phenolate ion stabilised by the keto-enol
form of the open-chain [Fig. 3 (inset)], by analogy to the proposed
Figure 1. ESMS spectra of HPLC-purified vitexin (A) and isovitexin (B) from crude D. uncinatum leaf protein incubated with UDP-glucose and [20,30,50,60-2H4]-1.

5658
M. L. Hamilton et al. / Tetrahedron Letters 50 (2009) 5656–5659
Figure 2. ESMS spectra of HPLC-purified vitexin from crude D. uncinatum leaf protein incubated with UDP-glucose and (C) [20,30,50,60-2H4]-5 or (D) [20,30,50,60-2H4]-6.
OH
HO
OH
O
Protein binding pocket
OH
H
H
Naringenin chalcone
O
O
O
Chalcone isomerase
OH
OH OH
HO
HO
HO
O
O
HO
OH
OH
O
O
OH
HO
HO
HO
HO
H
OH
OR
OH
O
Naringenin
OH
O
Flavanone 2-hydroxylase
Vitexin
OH
OH
OH
HO
HO
O
O
O
OH
OH
OH
O
HO
HO
OH
OH HO
O
O
O
D. uncinatum
C-glycosyltransferase
OH
OH
HO
OH
OH
OH
O
OH
OH
OH
2-Hydroxynaringenin
Dehydratase
OH
Isovitexin
O
O
Apigenin
Figure 3. Biosynthetic pathway for C-glucosylflavonoid biosynthesis in D. uncinatum and (inset) putative mechanism via the keto-enol tautomer of 2-hydroxynaringenin (1).
ceae). This further supports a general biosynthetic pathway for C-
References and notes
glucyosylflavones in plants (Fig. 3).
1. Parker, C. Pest Mange. Sci. 2009, 65, 453–459.
2. Hassanali, A.; Herren, H.; Khan, Z. R.; Pickett, J. A.; Woodcock, C. M. Philos. Trans.
R. Soc. B 2008, 363, 611–621.
3. Khan, Z. R.; Pickett, J. A.; Wadhams, L. J.; Hassanali, A.; Midega, C. A. O. Crop
Prot. 2006, 25, 989–995.
Acknowledgements
4. Khan, Z. R.; Pickett, J. A.; van den Berg, J.; Wadhams, L. J.; Woodcock, C. M. Pest.
Manage. Sci. 2000, 56, 957–962.
5. Khan, Z. R.; Hassanali, A.; Overholt, W.; Khamis, T. M.; Hooper, A. M.; Pickett, J.
A.; Wadhams, L. J.; Woodcock, C. M. J. Chem. Ecol. 2002, 28, 1871–1885.
Rothamsted Research receives grant-aided support from the
Biotechnology and Biological Sciences Research Council (BBSRC)
and was funded through the BBSRC/DFID SARID initiative.

M. L. Hamilton et al. / Tetrahedron Letters 50 (2009) 5656–5659
5659
6. Hooper, A. M.; Hassanali, A.; Chamberlain, K.; Khan, Z.; Pickett, J. A. Pest
Manage. Sci. 2009, 65, 546–552.
7. Fujita, M.; Inoue, T. Yanugaku Zasshi 1979, 99, 165–171.
8. Wallace, J. W.; Alston, R. E. Plant Cell Physiol. 1966, 7, 699–700.
9. Wallace, J. W.; Grisebach, H. Biochim. Biophys. Acta 1973, 304, 837–841.
10. Kerscher, F.; Franz, G. Z. Naturforsch., C 1987, 42, 519–524.
11. Kerscher, F.; Franz, G. J. Plant Physiol. 1988, 132, 110–115.
12. Brazier-Hicks, M.; Evans, K. M.; Gershater, M. C.; Puschmann, H.; Steel, P. G.;
Edwards, R. J. Biol. Chem. 2009, 284, 17926–17934.
Incubation: Enzyme analysis was performed according to Brazierand Cole17
with modifications. Protein pellets were desalted in 0.2 M HEPES–NaOH, pH 8,
containing 2 mM DTT using Sephadex G-25 gel filtration chromatography (PD-
10 columns, GE Healthcare). Protein content was determined using Bradford
reagent according to the manufacturer’s instructions (Sigma). Protein extract
was diluted to 2 mg/mL in buffer and pre-incubated at 30 °C with 66
[20,30,50,60-2H4]-1, -5 or -6 for 3 min. The reaction was initiated by the addition
of 100 M UDP-glucose, stopped after 45 min by the addition of 0.3 M HCl
(250 l) and extracted with EtOAc, 1.5 mL/mg protein. The organic layer was
lM
l
l
13. Kawamoto, H.; Nakatsubo, F.; Murakami, K. Synth. Commun. 1996, 26, 534–551.
14. Hauteville, M.; Chadenson, M.; Chopin, J. Bull. Soc. Chim. Fr. 1975, 1803–1808.
15. Cho, H.; Beale, J. M.; Graff, C.; Mocek, U.; Nakagaka, A.; Omura, S.; Floss, H. B. J.
Am. Chem. Soc. 1993, 115, 12296–12304.
evaporated under N2 and re-suspended in MeOH.
HPLC: Samples were isolated on an ACE 5 AQ column (250 mm ꢁ 4.6 mm),
using a mobile phase comprising two solvents, solvent A (H2O–HCO2H 95:5)
and solvent B (MeOH). Samples were eluted with a series of linear gradients set
initially at 95:5 A:B (0 min), 85:15 (3 min), 75:25 (13 min), 70:30 (25 min),
45:55 (35 min), 45:55 (45 min), 5:95 (46 min), 5:95 (55 min) and 95:5
(60 min).
ESMS: Purified fractions were collected from the HPLC column, evaporated
under N2 and re-suspended in MeOH prior to analysis by ESMS using an
Autospec Ultima Mass Spectrometer [Waters Corp (Micromass UK)] equipped
with an electrospray ionisation source (ESI). Positive ion mode ESI-MS was
performed at 4 kV and 80 °C, with N2 gas as the desolvation and nebuliser gas
(350 L/h and 15/20 L/h). The solvent used as mobile phase for ESI-MS was
MeOH–H2O (1:1) with 2% AcOH.
16. Materials and methods: Protein isolation: D. uncinatum seeds were obtained
from Western Seed Company, Kitale, Kenya. Plants were grown in a glasshouse
with an 8-h dark/ 16-h light cycle with day and night temperatures of 20 and
18 °C in Rothamsted Standard Compost Mix. Crude protein extract was isolated
according to Brazier and Cole17 with some modifications. Leaves were
harvested, weighed, snap frozen in liquid N2 and ground to a powder with a
pestle and mortar. The powder was extracted on ice with 5 v/w of 0.1 M
HEPES–NaOH, pH 7.2, containing 2 mM dithiothreitol (DTT) and 0.025 g/mL of
polyvinylpolypyrrolidone (PVP). The mixture was filtered through muslin prior
to centrifugation at 8000g for 30 min. Protein was precipitated between 45–
65% (NH4)2SO4 saturation and the pellet recovered by centrifugation at 27000g
for 60 min. Protein pellets were frozen in liquid N2 and stored at ꢀ80 °C.
17. Brazier, M.; Cole, D. J.; Edwards, R. Phytochemistry 2002, 59, 149–156.
18. Mittler, M.; Bechtold, A.; Schulz, G. E. J. Mol. Biol. 2007, 372, 67–76.