Chiral molecular recognition on formation of a metalloanthocyanin: A supramolecular metal complex pigment from blue flowers of salvia patens

Article abstract of DOI:10.1002/1521-3773(20010302)40:5<894::AID-ANIE894>3.0.CO;2-8

Most blue flower color is the result of a metalloanthocyanin, a stoichiometric self-assembled supramolecular pigment consisting of six molecules of an anthocyanin, six molecules of an anthocyanin, six molecules of flavone, and two metal atoms. We have iso

Full text of DOI:10.1002/1521-3773(20010302)40:5<894::AID-ANIE894>3.0.CO;2-8

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by NMR spectroscopy and X-ray crystallography.[1, 3] Very
fine and strict molecular recognition,[5] including chiral
molecular recognition, occurs during reconstruction. The
aromatic chromophores in commelinin and protocyanin stack
in a chiral manner, with anticlockwise self-association be-
tween two molecules of the anthocyanin or two molecules of
the flavone, and clockwise stacking of anthocyanidin and
flavone nuclei (co-pigmentation).[3] These phenomena must
arise from the chirality of the sugars attached to the
anthocyanidin and flavone units, although, no evidence has
hitherto been provided in support of this conclusion.
mica plate touched the drop before the folding of the surface was
established (Figure 2b). The aqueous solution in the cuvette was removed
by using a Hamilton pump and the solvent inside was evaporated. The
resulting film was studied by surface force microscopy. A similar process
was used for the collapsed drop (Figure 2 f).
Received: November 4, 1999
Revised: September 14, 2000 [Z14230]
[1] M. Bos, T. Nylander, T. Arnebrant, D. C. Clark, Food Emulsifiers and
Their Applications (Eds.: G. L. Hasenhuettl, R. W. Hartel), Chapman
and Hall, New York 1997, pp. 95 ± 146.
Herein we describe the chiral molecular recognition on
formation of the metalloanthocyanin protodelphin (1).[6]
Elucidation of the gross structure of 1, a genuine pigment
from blue petals of Salvia patens (Figure 1), demonstrated
[2] a) J. B. Li, V. B. Fainerman, R. Miller, Langmuir 1996, 12, 5138 ± 5143;
b) J. B. Li, H. Chen, J. Wu, J. Zhao, R. Miller, Colloids Surf. B 1999, 15,
289 ± 295.
[3] M. A. Bos, T. Nylander, Langmuir 1996, 12, 2791 ± 2797.
[4] H. Clausen-Schaumann, M. Grandbois, H. E. Gaub, Adv. Mater. 1998,
10, 950 ± 952.
[5] V. B. Fainerman, J. Zhao, D. Vollhardt, A. V. Makievski, J. B. Li, J.
Phys. Chem. B 1999, 103, 8998.
[6] J. B. Li, R. Miller, H. Möhwald, Colloids Surf. A 1996, 114, 113 ± 121.
[7] U. Dahmen-Levison, G. Brezesinski, H. Möhwald, Prog. Colloid
Polym. Sci. 1998, 110, 269 ± 274.
[8] E. Donath, G. B. Sukhorukov, F. Caruso, S. A. Davis, H. Möhwald,
Angew. Chem. 1998, 16, 2324 ± 2326; Angew. Chem. Int. Ed. 1998, 16,
2201 ± 2205.
Figure 1. Salvia patens.
Chiral Molecular Recognition on Formation of
a Metalloanthocyanin: A Supramolecular
Metal Complex Pigment from Blue Flowers of
Salvia patens**
that it consists of the anthocyanin malonylawobanin (2),[7] the
flavone apigenin 7,4'-di-O-b-d-glucoside (3a),[6, 8] and Mg2‡
ions (Scheme 1). General and effective synthetic procedures
for anthocyanins and/or flavones containing the optically
antipodal glycosides are necessary for chiral recognition
studies. We have developed a reliable glycosylation method
for less-reactive phenols in flavonoids utilizing a new Lewis
acid/base promoted glycosylation[9] with the peracetylglucosyl
fluoride (4),[10] and succeeded in preparing natural apigenin
7,4'-di-O-b-glucoside 3a and unnatural derivatives (partly)
substituted with l-glucose (3b ± d). Employment of these
synthetic apigenin derivatives and natural 2 in the presence of
Mg2‡ ions allowed an examination of the chiral molecular
recognition that occurs during formation of the blue complex
pigment.
Tadao Kondo,* Kin-ichi Oyama, and Kumi Yoshida
Most blue flower color is the result of a metalloanthocya-
nin,[1, 2] a stoichiometric self-assembled supramolecular pig-
ment consisting of six molecules of an anthocyanin, six
molecules of a flavone, and two metal atoms.[3, 4] We have
isolated various metalloanthocyanins from the petals of blue
flowers and elucidated their structures. We have clarified the
mechanisms of the development of blue color by chemical
reconstruction of some supramolecules and structural analysis
For the structural determination,[11] 1 was synthesized from
the components according to our reconstruction method.[3]
Compounds 2 and 3a and Mg2‡ ions were mixed in a weakly
alkaline solution, then the mixture was purified by gel-
permeation chromatography/liquid chromatography (GPC-
LC)[12] to give pure protodelphin (1) in 61% yield. The UV/
Vis spectra of the product were completely identical with
those of the naturally occurring form[6] (Figure 2). Electro-
spray ionization mass spectrometry (ESI-MS)[13] of 1 gave a
multiply charged molecular ion at m/z: 1751.8 ([M 5H]5 )
corresponding to the constitution C396H408O222Mg2 (average
molecular weight: 8767.95, calcd 1751.37 [M 5H]5 ). The
[*] Prof. Dr. T. Kondo, K.-i. Oyama
Chemical Instrument Center
Nagoya University
Furo-cho, Chikusa, Nagoya 464-8602 (Japan)
Fax : (‡81)52-789-3062
E-mail: tkmail@cic.nagoya-u.ac.jp
Prof. Dr. K. Yoshida
Graduate School of Human Informatics
Nagoya University
Furo-cho, Chikusa, Nagoya 464-8601 (Japan)
[**] We thank Mr. Y. Maeda (Chemical Instrument Center, Nagoya
University) for help with molecular modeling, and the Ministry of
Education, Science, Sports, and Culture of Japan (COE Research
No. 07CE2004) for financial support.
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Figure 2. UV/Vis and CD spectra of reconstructed mixtures of
2
(5Â10 4 m) and 3, 9, or 10a (1 equiv) in the presence of Mg2‡ ions
(5 equiv) in phosphate buffer (pH 6.0). Iˆ2‡3a‡Mg2‡
,
IIˆ
2‡3b‡Mg2‡, IIIˆ2‡3c‡Mg2‡, IVˆ2‡3d‡Mg2‡, Vˆ2‡9‡Mg2‡
,
VIˆ2‡10a‡Mg2‡
.
We achieved the first synthesis of apigenin 7,4'-di-O-b-d-
glucoside (3a) by direct glycosylation of the flavone nucleus
(Scheme 2). Condensation of naringenin (5) with peracetyl-d-
glucosyl bromide (6a) and consequent oxidization with DDQ
OH
OH
HO
O
O
RO
O
O
Scheme 1. The structure of components (top and middle) and the entire
(bottom) protodelphin. M (2) as a bidentate ligand is coordinated to Mg2‡
ions, and F (3a) is intercalated.
a, b
OH
OH
5
7a, b
CD spectrum (Figure 2) showed a strong negative exciton-
type Cotton effect around 620 nm, which indicates that the
anthocyanidin nuclei must be closely stacked in an anticlock-
wise manner, and the 1H NMR spectrum showed simple
signals attributable to one anthocyanin and one flavone
residue. Analysis of long-range NOEs between protons of 2
and those of 3a suggested a cross-parallel arrangement of
nuclei. These data indicate that the arrangement of the
components in the gross structure of 1 is very similar to that of
commelinin. Thus, protodelphin is a stoichiometric supra-
molecular metal complex pigment, that is, a metalloantho-
cyanin, similar to commelinin and protocyanin (Scheme 1).
Although glycosylflavones are widely distributed in plants
and have attracted attention because of their important
biological and medical aspects, only a few syntheses of poly-
O-glycosylflavones with satisfactory yield have, surprisingly,
been reported. The nucleophilicity of the 4'-hydroxy group on
the B-ring of the flavone nucleus is very poor, therefore, there
are very few examples of direct 4'-O-glycosylation.[14]
OR'
RO
O
O
c
d
3a-d
OH
8a-d
OAc
AcO
O
O
R2 =
OAc
OAc
AcO
AcO
R1 =
OAc
AcO
Scheme 2. Synthesis of apigenin 7,4'-di-O-b-glucosides 3: a) peracetylglu-
cosyl bromide (6a or 6b), Ag2CO3, quinoline; b) DDQ, 1,4-dioxane, 7a:
R ˆ R1 (66%), 7b: R ˆ R2 (66%); c) peracetylglucosyl fluoride (4a or 4b),
BF3 ´ Et2O, DTBMP/TMG (4/1), CH2Cl2/PhCl (1/6), 8a: R ˆ R' ˆ R1
(70%), 8b: R ˆ R' ˆ R2 (72%), 8c: R ˆ R1, R' ˆ R2 (66%), 8d: R ˆ R2,
R' ˆ R1 (68%); d) 1. NaOMe, MeOH/CHCl3 (2/1); 2. Dowex 50W-8X
‡
(H ), 3a (92%), 3b (88%), 3c (92%), 3d (94%). 4a, 6a: d-glucosyl; 4b,
6b: l-glucosyl. DDQˆ 2,3-dichloro-5,6-dicyanobenzoquinone; DTBMPˆ
2,6-di-tert-butyl-4-methylpyridine; TMG ˆ 1,1,3,3-tetramethylguanidine.
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gave the apigenin 7-O-peracetyl-b-d-glucoside (7a). Treat-
ment of 7a with peracetyl-d-glucosyl fluoride (4a) in the
presence of BF3 ´ OEt2, DTBMP, and TMG afforded apigenin
7,4'-O-di-b-d-glucoside as an acetate[14b] (8a) in 70% yield,
which was deprotected to yield 3a. A combination of DTBMP
and TMG was essential for the glycosylation of the 4'-OH
group of 7a. By using the same procedure we could prepare a
series of unnatural apigenin 7,4'-di-O-b-glucosides (3b ± d)
derived from l-glucose: the 7,4'-di-l-glucoside 3b, 7-d-gluco-
side-4'-l-glucoside 3c, and 7-l-glucoside-4'-d-glucoside 3d.
To clarify the role of the flavone part in the chiral
recognition on formation of the metalloanthocyanin, we
mixed 2 with 3b ± d in the presence of Mg2‡ ions. Among
the three unnatural apigenin diglucosides used, only 3d gave a
blue solution (Figure 3, IV); 3b and 3c gave purple solutions
(II and III, respectively). Solution IV showed almost the same
UV/Vis and CD spectra as protodelphin (Figure 2), which
indicates that a protodelphin-like metalloanthocyanin was
produced from 3d. The stability of the colors of those
mixtures was recorded and the results are shown in Figure 3.
The color of 1 in a neutral aqueous solution (I) was very
stable, while the blue color of IV was less so. The purple color
of II and III rapidly faded. To reveal the role of the glucosides
at the 7- and 4'-OH groups of apigenin, we prepared apigenin
residue at the 4'-OH position is indispensable for formation of
a metal complex pigment. The d-glucose residue at the 7-OH
position could stabilize the molecular association, while an l-
glucose would destabilize the complex by steric hindrance.
To estimate the enantioselectivity of the molecular recog-
nition on formation of the supramolecule, we attempted a
reconstruction experiment from 2 and a 1:1 mixture of 3a and
3b with Mg2‡ ions. The blue mixture was purified by GPC-LC,
and a blue-black amorphous mass was obtained in 85% yield.
The ratio of 3a to 3b in the supramolecule was analyzed by
HPLC on a chiral stationary phase.[17] No peak corresponding
to 3b was observed in the chromatogram, thus the ratio of 3b
to 3a is less than 2:98.[18] This result indicated that l,l-
diglucoside 3b was completely excluded from the metal
complex. The natural anthocyanin molecule chose only the d-
series of glycosylflavones as partners to form metalloantho-
cyanin.
The high enantioselectivity observed during formation of
the supramolecule could be caused by the chiral stacking
arrangement of components in 1 as a consequence of the
presence of the sugar moiety. Three flavone molecules[19] in 1
associate to form a M (minus) helical structure, similar to a
propeller with three blades. They are bound at the pivot point
by a strong hydrogen-bonding network among the hydroxyl
groups at C-2 and C-3 of the 4'-O-b-d-glucopyranosides
(Figure 4). The two sets of M-helical flavone associates fit
closely into the vacant space formed from the metal complex
of six molecules of 2 and two Mg2‡ ions. Replacement of d- by
l-glucose at the 4'-OH position of apigenin could invert the
helicity to obtain the P (plus) form, with the consequence that
the associates of the flavones 3b, 3c, and 10b do not fit into
the vacant space. Thus, the M helicity formed by the three
molecules containing the d-glucosyl residue at the 4'-OH site
plays a key role in the formation of metalloanthocyanin.
In conclusion, malonylawobanin (2) chooses only the
d chirality of the 4'-O-glucosyl residue in apigenin 7,4'-di-
glucoside to form the stoichiometric supramolecular metal
complex pigment protodelphin. This restricted chiral and
structural recognition controls the entire self-assembly of the
metalloanthocyanin and is responsible for the beautiful blue
color of the flowers.
Experimental Section
Figure 3. Color stabilities of mixtures of 2 (5 Â 10 4 m) and 3, 9, or 10a
(1 equiv) in the presence of Mg2‡ ions (5 equiv) in phosphate buffer
(pH 6.0). I ˆ 2 ‡ 3a ‡ Mg2‡, II ˆ 2 ‡ 3b ‡ Mg2‡, III ˆ 2 ‡ 3c ‡ Mg2‡, IVˆ
Reconstruction of protodelphin (1): Caution: the reaction requires
concentrations of at least 10 2 ± 10 3 m. A solution of 3a (20 mg) in water
(1 mL) and 0.5m Mg(OAc)2 (0.2 mL) was added under stirring at RT to a
solution of 2 (34 mg, 35 mmol) in water (0.6 mL) which was neutralized with
1.3% aqueous ammonia. The resulting blue solution was separated by
2 ‡ 3d ‡ Mg2‡, Vˆ 2 ‡ 9 ‡ Mg2‡, VI ˆ 2 ‡ 10a ‡ Mg2‡
.
7-O-b-d-glucoside[15] (9) and 4'-O-b-d-glucoside[16] (10a),
respectively, and examined whether a protodelphin-like
supramolecule was formed. Compound 10a gave the blue
pigment with 2 and Mg2‡ ions, but 9 did not (Figure 2, 3).
Surprisingly, the blue color of VI, constructed from 10a, was
more stable than that of IV produced from 3d. Furthermore,
we checked its enantiomer, apigenin 4'-O-b-l-glucoside
(10b), for complex formation but no metalloanthocyanin
was generated. These phenomena indicate that the d-glucosyl
GPC-LC (Cellurofine GC-15-m) to give 1 as a blue-black mass (34 mg,
1
~
~
*
*
61%). H NMR (600 MHz, D2O; for
,
,
, and see Scheme 1): d ˆ 4.53
*
(brd, J ˆ 7.5 Hz, 1H; -1), 4.77 (brs, 1H; M-8), 4.87 (brd, J ˆ 7.5 Hz, 1H;
~
~
*
-1), 4.94 (brd, J ˆ 7.5 Hz, 1H; -1), 5.13 (brd, J ˆ 7.5 Hz, 1H; -1), 5.31
(brs, 1H; F-8), 5.52 (brs, 1H; F-3), 5.62 (brs, 1H; M-6), 5.82 (d, J ˆ 16 Hz,
1H; M-a), 5.98 (brs, 1H; F-6), 6.44 (3H; M-3'', 5'', F-2'), 6.62 (brd, 2H;
F-2', 3'), 6.73 (1H; M-4), 6.35 (d, J ˆ 9 Hz, 2H; M-2'',6''), 6.75 (d, J ˆ 16 Hz,
1H; M-b), 5.62 (s, 1H; F-3), 7.46 (brs, 2H; F-5',6'), 7.79 (brs, 1H; M-6'), 8.10
(brs, 1H; M-2'); MS (ESI): m/z: 1751.8 [M 5H]5
.
Received: August 15, 2000 [Z15638]
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[8] N. C. Veitch, R. J. Grayer, J. L. Irwin, K. Takeda, Phytochemistry 1998,
48, 389 ± 393.
[9] Our glycosylation procedure (K.-i. Oyama, T. Kondo, Synlett 1999,
1627 ± 1629) was modified.
[10] M. Hayashi, S. Hashimoto, R. Noyori, Chem. Lett. 1984, 1747 ± 1750.
[11] The composition and gross structure of natural 1 could not be clarified
yet because of difficulty in purification. Complex 1 is soluble only in
aqueous media and upon dilution quickly dissociates and becomes
decolorized.
[12] The pigment was eluted in the void fraction of GPC (Cellurofine GC-
15-m) with H2O.
[13] T. Kondo, M. Ueda, K. Yoshida, K. Titani, M. Isobe, T. Goto, J. Am.
Chem. Soc. 1994, 116, 7457 ± 7458.
Â
Â
[14] a) L. Farkas, A. Wolfner, M. Nogradi, H. Wagner, L. Hörhammer,
Chem. Ber. 1968, 101, 1630 ± 1632; b) C. Demetzos, A.-L. Skaltsounis,
F. Tillequin, M. Koch, Carbohydr. Res. 1990, 207, 131 ± 137.
[15] 7a was treated with NaOCH3/CH3OH to give apigenin 7-O-b-d-
Â
Â
glucoside (9); M. Nogradi, L. Farkas, H. Wagner, L. Hörhammer,
Chem. Ber. 1967, 100, 2783 ± 2790.
[16] Apigenin 4'-O-b-d- and -l-glucoside (10a,b) were prepared according
to our procedure. Naringenin (5) was selectively silylated at the 7-OH
position with tert-butyldimethylsilyl chloride/imidazole in DMF. The
resulting 7-O-silylnaringenin was oxidized, glycosylated with perace-
tyl-d- or l-glucosyl fluoride, and deprotected with tetrabutylammo-
nium fluoride (TBAF) and MeONa/MeOH to provide 10a,b.
[17] Aqueous trifluoroacetic acid (TFA) was added to the produced
pigment, then the solution was analyzed by HPLC using a CHIR-
ALCEL OD-R column (DAICEL CHEMICAL) eluted with TFA/
CH3CN/water 0.3/13/86.7 at RT.
[18] A mixture of 3a and 3b (ratio 98:2) gave two peaks completely
separated in HPLC.
[19] The starting structure of 1 was built by changing the flavocommelin
molecule to 3a on the basis of the crystallographic structure of
commelinin.[3] The local optimization of this structure was performed
by QUANTA-97-CHARMm (Ver. 23.2) software.
Synthesis of an Array Comprising 837 Variants
of the hYAP WW Protein Domain**
Figure 4. The optimized M-helical conformer of three 3a molecules,
similar to a propeller with three blades (top), and the gross structure of 1
(bottom). One set of three F molecules is located at the upper side of the
pigment and one at the lower side. The F molecules are intercalated in a
face-to-face manner. Mg2‡ ions exist at both the upper and lower part of the
superstructure. The hydrogen atoms are omitted in 1 for clarity. Color
scheme: anthocyanin 2: blue, flavone 3a: yellow, and Mg2‡: red.
Â
Florian Toepert,* Jose R. Pires, Christiane Landgraf,
Hartmut Oschkinat, and Jens Schneider-Mergener*
The elucidation of structure ± function relationships of
proteins contributes to a better understanding of how they
work and also provides clues for the synthesis of agonists and
antagonists. Today, variants of the investigated protein
required for structure ± function analyses are produced almost
[1] T. Goto, T. Kondo, Angew. Chem. 1991, 103, 17 ± 33; Angew. Chem.
Int. Ed. Engl. 1991, 30, 17 ± 33.
[*] Dipl.-Biochem. F. Toepert, Prof. Dr. J. Schneider-Mergener,[‡]
C. Landgraf
[2] a) K. Hayashi, Y. Abe, S. Mitsui, Proc. Jpn. Acad. 1958, 34, 373 ± 378;
b) K. Hayashi, K. Takeda, Proc. Jpn. Acad. 1970, 46, 535 ± 540; c) K.
Hayashi, N. Saito, S. Mitsui, Proc. Jpn. Acad. 1961, 37, 393 ± 397.
[3] a) T. Kondo, K. Yoshida, A. Nakagawa, T. Kawai, H. Tamura, T. Goto,
Nature 1992, 358, 515 ± 518; b) T. Kondo, M. Ueda, M. Isobe, T. Goto,
Tetrahedron Lett. 1998, 39, 8307 ± 8310.
[4] We have revealed that the blue pigment from Salvia uliginosa and
Nemophila menziesii is a metalloanthocyanin by analysis of the
natural pigment and from components reconstructed ones; T. Kondo,
K.-i. Oyama, K. Yoshida, unpublished results.
Institut für Medizinische Immunologie
Â
Charite, Humboldt-Universität Berlin
Schumannstrasse 20 ± 21, 10098 Berlin (Germany)
Fax : (‡49)030-2802-6460
E-mail: florian.toepert@charite.de, jsm@charite.de
Dr. J. R. Pires, Prof. Dr. H. Oschkinat
Forschungsinstitut für Molekulare Pharmakologie
Robert-Rössle-Strasse 10, 13125 Berlin (Germany)
‡
[ ] Current address:
[5] T. Kondo, K. Yoshida, M. Yoshikane, T. Goto, Agric. Biol. Chem. 1991,
55, 2919 ± 2921.
[6] K. Takeda, M. Yanagisawa, T. Kifune, T. Kinoshita, C. F. Timberlake,
Phytochemistry 1994, 35, 1167 ± 1169.
Jerini AG
Rudower Chaussee 29, 12489 Berlin (Germany)
[**] This work was supported by the DFG (INK 16/B1-1), by the Fonds der
Â
Chemischen Industrie, and by the Universitätsklinikum Charite
Berlin.
[7] T. Goto, T. Kondo, H. Tamura, S. Takase, Tetrahedron Lett. 1983, 24,
4863 ± 4866.
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