Application of a regioselective Mannich reaction on naringenin and its use in fluorescent labeling

Article abstract of DOI:10.1055/s-2006-941564

A novel strategy for site-specific fluorescent labeling on naringenin (1) was established by a new direct Mannich reaction in combination with a Huisgen [3+2]-cycloaddition reaction. High regioselectivity was observed for direct Mannich reactions on narin

Full text of DOI:10.1055/s-2006-941564

LETTER
1225
Application of a Regioselective Mannich Reaction on Naringenin and its Use in
Fluorescent Labeling
Regioselective
M
e
annichR
i
eactionand
F
C
luorescent
L
abe
l
h
ingof Naring
e
enin n, Tai-Shan Hu, Jing Zhu, Houming Wu, Zhu-Jun Yao*
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, P. R. of China
Fax +86(21)64166128; E-mail: yaoz@mail.sioc.ac.cn
Received 2 December 2005
potential site-selective derivation protocol for naringenin,
Abstract: A novel strategy for site-specific fluorescent labeling on
naringenin (1) was established by a new direct Mannich reaction in
combination with a Huisgen [3+2]-cycloaddition reaction. High
regioselectivity was observed for direct Mannich reactions on
which could introduce sites for fluorescent labeling
through further elaboration. Other positions in naringenin,
such as the a-carbon of the carbonyl on the B ring and the
naringenin and several other flavonones using a variety of amines C-2¢ of the C ring could also potentially be involved in
and aldehydes.
such Mannich reactions (Figure 1). Previous studies on
electrophilic reactions of naringenin under a variety of
conditions indicates that mixtures often result through
substitution at different positions, including C-6 and C-8
of the A ring and C-2¢ of the C ring, as well as C-3 of the
B ring.8–12 In order to acquire pure single Mannich
adducts, one frequently adopted approach is to protect one
or more phenolic hydroxyls prior to the Mannich reac-
tions. However, such an indirect approach usually re-
quires multi-step transformations. Obviously, it could be
of great value to find a regioselective Mannich reaction,
which would allow the addition of extended functional-
ized alkyl groups to naringenin without the need for pro-
tecting groups. Such a process would be of great utility in
the study of phenolic flavonones. Though the phenolic
hydroxyl groups in naringenin are chemically similar, fine
differences exist in the chemical and electronic environ-
ments of each. After reviewing the literature of other com-
pounds with multiple phenolic hydroxyl groups, we
expected the C-6 of naringenin to be the most reactive site
for electrophillic reactions.13 Optimization of appropriate
Mannich conditions may provide a highly regioselective
facile entrance to naringenin derivatives. Herein, we
describe our efforts to achieve such a reaction.
Key words: naringenin, Mannich reaction, Huisgen cycloaddition,
regioselectivity, fluorescent labeling
Naringenin (1, Figure 1) is a representative flavonone
existing in many plants and fruits (e.g. grapefruit). Recent
studies show that naringenin protects against toxins that
are found in chemotherapy drugs and some environmental
sources. It also enhances lipid and ethanol metabolism, as
well as serving as a free-radical scavenger and an anti-
oxidant.1 More interestingly, such flavonones have also
been shown to play an important role in nitrogen fixation
in a variety of legume species. Many studies have shown
that naringenin is an active small molecule involved in the
control of root nodulation, which is a prerequisite for
nitrogen fixation. The interaction between naringenin,
which is released by the plant, with nod-D protein induces
the over-expression of nod genes and leads to the produc-
tion of NodRm-IV (sulfated lipooligosaccharides), which
are Rhizobium nodulation signaling molecules. During
nodulation of legumes, the interaction between narigenin
and nod-D protein is the first important step.2 Although
genetics show that nod-D and naringenin interact with
each other, there is no direct evidence proving this.3,4 In
order to unambiguously demonstrate direct interactions
between nod-D and naringenin, suitable naringenin deriv-
atives containing fluorescent labels are required for visu-
alization techniques such as fluorescence resonance
energy transfer (FRET).5 Herein, we report the results of
our studies on direct Mannich reactions of unprotected
naringenins, as well as their application to the above-men-
tioned efforts to fluorescently label naringenin deriva-
tives.
OH
O
5
4
10
6
7
3
2
A
B
3'
C
2'
2'
1'
HO
O
1
9
4'
3'
8
OH
naringenin (1)
Figure 1 Structure of naringenin (1) and possible sites for Mannich
reaction
Structural analysis indicates that the A ring of naringenin,
especially positions C-6 and C-8, might be more reactive
towards electrophiles than the C ring (Figure 1). There-
fore, relatively mild Mannich reactions6,7 might provide a
N-Methyl b-alanine methyl ester (Table 1, 3g) was chosen
as the first amine substrate in combination with paraform-
aldehyde to find appropriate conditions for a selective
Mannich addition. Fortunately, success was achieved
after investigating a variety of conditions. Initial studies
showed that temperature and duration of the reaction
significantly affected the yield of the major product. No
SYNLETT 2006, No. 8, pp 1225–1229
1
5.
0
5.
2
0
0
6
Advanced online publication: 05.05.2006
DOI: 10.1055/s-2006-941564; Art ID: W32505ST
© Georg Thieme Verlag Stuttgart · New York

1226
L. Chen et al.
LETTER
reaction was observed at room temperature in ethanol. Mannich adducts (C-6 vs C-8, 4:1) was obtained when the
Only a highly polar product (difficult to purify) was af- reaction was carried out at 65 °C (Table 3, entry 1); a sin-
forded when the reaction was heated to reflux for one gle product (2a, C-6 adduct) was afforded in 77% isolated
hour. However, when the reaction was stirred at 50 °C for yield (Table 3, entry 2) when the reaction was performed
16 hours, a single Mannich product 2g was generated in at 55 °C. To our knowledge, this is the first demonstration
61% isolated yield. To better understand this reaction, N- that a direct Mannich reaction on naringenin without any
methylbenzylamine (3a) was selected as the secondary protecting groups can be well controlled to afford a single
amine substrate to replace N-methyl b-alanine methyl product regioselectively.
ester (3g). Similarly, temperature was found to be an im-
portant factor affecting the regioselectivity. A mixture of
combinations were examined under similar conditions. As
A variety of secondary amines and aldehydes in various
Table 1 Mannich Reactions of Naringenin with Secondary Amines and Aldehydes
R1 OH
O
OH
O
R1CHO
R2R3NH (3)
R2
N
R3
O
H
O
HO
O
OH
OH
product (2)
naringenin (1)
Entry
1
Aldehyde
(HCHO)n
Amine
Conditions
Products
Yieldsa
77%
2 h, 55 °C
2 h, 55 °C
2a: R1 = H; R2 = Me; R3 = Bn
NH
3a
O
2
(HCHO)n
2b: R1 = H; R2,R3 = -(CH2CH2OCH2CH2)-
80%b
N
H
3b
HN
3
4
5
(HCHO)n
(HCHO)n
(HCHO)n
24 h, 60 °C
8 h, 45 °C
3 h, 60 °C
2c: R1 = H; R2 = R3 = Et
66%
68%
90%c
3c
HN
2d: R1 = H; R2 = Me; R3 = CH2C≡CH
2e: R1 = H; R2 = Me; R3 = (CH2)6N3
3d
H
N
N3
3e
OH
O
O
NH
HN
6
(HCHO)n
24 h, 60 °C
72%c,d
HO
OH
3f
2f
H
O
N
7
8
(HCHO)n
(HCHO)n
16 h, 50 °C
8 h, 20 °C
2g: R1 = H; R2 = Me; R3 = CH2CH2CO2Me
2h: R1 = H; R2 = Me; R3 = CH2CH2CO2-t-Bu
61%
O
O
3g
3h
H
N
66%e
O
O
9
CH3CHO
7 h, 35 °C
2i: R1 = CH3; R2,R3 = -(CH2CH2OCH2CH2)-
75%
N
H
3b
a Isolated yields.
b Structure of 2b was determined by single-crystal X-ray analysis.
c Reagents: ZnCl2 (0.1 equiv), EtOH, 60 °C.
d Two by-products, 4a and 4b, were obtained.
e Structure of 2h was confirmed by 2D-NMR studies (HMQC and HMBC).
Synlett 2006, No. 8, 1225–1229 © Thieme Stuttgart · New York

LETTER
Regioselective Mannich Reaction and Fluorescent Labeling of Naringenin
1227
the results show (Table 1), all reactions, irrespective of
electrophiles, performed well under mild conditions, to
afford predominantly the C-6 substituted derivatives.
These products are very polar and easily absorbed onto
silica gel with moderate isolated yields obtained in most
cases following chromatographic purification. The
regioselectivities and chemical yields of the naringenin
Mannich reactions can be optimized by changing the reac-
tion temperatures and reaction times (Table 1).
OH
O
O
NH
N
H
HO
OH
N
N
H
H
4a
4b
Figure 2 By-products of the Mannich reaction of naringenin with
N-methylaniline and paraformaldehyde
With the exception of N-methylaniline (3f), all secondary
amines gave single products. In the case of N-methyl-
aniline (Table 1, entry 6), the reaction occurred at the
para-position of the phenyl ring under the general reac-
tion conditions, affording 2f in 12% yield along with two
by-products 4a and 4b (Figure 2).14 This might be due to
the higher electron density at the para-position of the
benzene ring. To improve the yield of this reaction, we
also tried the addition of Lewis acids and other solvents
(e.g. CH3COOH–H2O, 1:1 or 1:3; DMF; EtOH, cat. HCl;
AlCl3, CH2Cl2). The most efficient conditions were found
to be 0.1 equivalent of ZnCl2 in ethanol at 60 °C, which
increased the chemical yield to 72%. Such conditions also
can be applied to the Mannich reaction of secondary
amine 3e, which gave no reaction either at room tempera-
ture or by heating; addition of 0.1 equivalent of ZnCl2
greatly improved the reaction (Table 1, entry 5) and
afforded the corresponding adduct 2e in 90% yield.
Significant temperature effects on the regioselectivity (C-
6 vs. C-8) were observed in Mannich reactions of narin-
genin with three secondary amines (Table 3). Reaction
with N-methyl-N-propargylamine (3d) and paraformalde-
hyde gave C-6 and C-8 adducts in almost equal amounts
at 65 °C, while a 50:1 ratio (C-6 vs C-8) resulted when the
reaction was performed at 45 °C (Table 3, entry 3 and 4).
Results with the other two amines were also in good
agreement with the above observations. These results
demonstrate that the A ring of naringenin is the most elec-
tron-rich with the C-6 position being the predominant site
for Mannich reactions.13
The direct and highly regioselective Mannich reactions
described above afford a facile entry to bio-labeling nar-
ingenin derivatives with fluorescent groups. With azido
derivative 2e in hand, a Huisgen [3+2] cycloaddition was
devised as the key step for coupling with the fluorescein
derivative 9 (Scheme 1) to generate the corresponding
fluorescent compound 10.
Two additional flavonones, 5 and 7, with structural fea-
tures similar to naringenin (1), were also examined under
these reaction conditions (Table 2). Both gave similar
regioselectivities favoring the C-6 position (6 and 8, re-
spectively).
Table 2 Mannich Reactions of Naringenin-Like Flavonoids with Paraformaldehyde and Morpholinea
Entry
1
Flavonoid
Conditions
55 °C, 2 h
Products
Yields
80%b
OH
O
O
OH
O
O
N
O
HO
HO
OH
OH
1
2b
OH
O
O
OH
O
O
N
O
2
3
40 °C, 3 h
79%b
HO
HO
5
6
OH
O
O
OH
O
O
OH
OH
N
O
OH
OH
OH
OH
r.t., 4 h
90%c
HO
HO
7
8
a Reaction conditions: flavonoid (1 mmol), paraformaldehyde (1.5 mmol), morpholine (1.5 mmol), EtOH (4 mL).
b Isolated yields following purification by silica gel column chromatography.
c Crystallization from H2O as the hydrochloride salt.
Synlett 2006, No. 8, 1225–1229 © Thieme Stuttgart · New York

1228
L. Chen et al.
LETTER
Table 3 Effects of Temperature on the Regioselelctivity of Mannich Reactions
OH
8
O
O
OH
O
O
OH
8
O
O
R1
6
(HCHO)n
R1R2NH
6
N
R2
H
O
R1
O
H
HO
EtOH
N
R2
OH
OH
OH
A
B
naringenin (1)
Entry
R1R2NH
3a
Temperature
65 °C
Yielda
68%
77%
30%
68%
20%
66%
Ratio (A/B)b
4:1
1
2
3
4
5
6
3a
55 °C
A only (2a)
1:1
3d
65 °C
3d
45 °C
50:1 (2d, major product)
ca. 50:1
3c
75 °C
3c
60 °C
A only (2c)
a Isolated yields.
b Measured by 1H NMR spectroscopy.
ative 9 was prepared from commercially available fluo-
rescein in two steps (Scheme 2). Treatment of fluorescein
(11) with excess propargyl bromide in the presence of
K2CO3 gave 1215 in quantitative yield. All initial efforts at
selective and/or direct preparation of 9 resulted in mix-
tures which could not be purified efficiently. Hydrolysis
of propargyl ester 12 was carried out smoothly using 1 M
LiOH in THF–H2O (1:1), affording 916 in excellent yield.
The ‘click’ reaction17,18 between azido-naringenin deriva-
tive 2e and the acetylene-fluorescein derivative 9 was
accomplished in t-BuOH and H2O using CuSO4·5H2O as
a catalyst and ascorbic acid to afford the fluorescein-
labeled naringenin 10 in 67% yield.19 UV analysis of
10 (in MeOH) indicated typical fluorescein absorptions
at 453 nm (e = 15967 M–1cm–1) and 478 nm (e = 13271
M–1cm–1) in addition to the naringenin absorptions at 279
nm and 324 nm. Fluorescence characterization of 10
shows lem at 523 nm (F = 0.20, in MeOH, using lexc 478
nm and quinine sulfate as a standard).
HO
N3
O
O
OH
O
O
OH
+
N
O
O
2e
OH
9
Huisgen
[3+2] cycloaddition
OH
O
N
N
HO
O
N
N
OH
O
O
OH
O
O
10
Scheme 1 Naringenin fluorescent-labeling strategy utilizing
Mannich adduct 2e.
O
O
O
In summary, direct Mannich reactions onto naringenin
were investigated without the use of phenolic protection.20
High regioselectivity for the C-6 position achieved under
optimized conditions indicated that electronic density in
the naringenin A ring could be exploited. Further studies
on related substrates showed such regioselectivity could
also be achieved under similar conditions. Based on
Mannich adduct 2e, a quick and convenient fluorescent
labeling strategy for naringenin was established, affording
the first fluorescein-labeled naringenin derivative 10.
Further biologically relevant studies with nod-D protein,
utilizing this fluorescently labeled compound are current-
ly underway and will be reported in due course. In addi-
tion, the Mannich adduct 2d having terminal acetylene
functionality offers alternate fluorescent labeling options
using ‘click’ chemistry. This work is also under investiga-
tion in our laboratory.
HO
O
O
a
O
O
COOH
12
fluorescein (11)
O
O
OH
c
b
10
2e
O
O
9
Scheme 2 Reagents and conditions: a) 3-bromopropyne, K2CO3,
DMF, 100%; b) 1 M LiOH, THF–H2O (1:1), 95%; c) 2e,
CuSO4·5H2O (cat.), ascorbic acid, t-BuOH, H2O, 67%.
Chemically stable acetylene-terminated fluorescein deriv-
Synlett 2006, No. 8, 1225–1229 © Thieme Stuttgart · New York

LETTER
Regioselective Mannich Reaction and Fluorescent Labeling of Naringenin
1229
(15) Berscheid, R.; Nieger, M.; Voegtle, F. Chem. Ber. 1992,
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Acknowledgment
This work was financially supported by the Major State Basic
Research and Development Program (2002CB0713808), NSFC
(20425205 and 20321202), Chinese Academy of Sciences
(KGCX2-SW-209), and Shanghai Municipal Commission of
Science and Technology (04DZ14901). The authors thank Dr.
Terry Burke at NIH, Maryland for helpful comments.
(18) Some recent examples of ‘click’ chemistry: (a) Lewis, W.
G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.;
Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int.
Ed. 2002, 41, 1053. (b) Wang, Q.; Chan, T. R.; Hilgraf, R.;
Folin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc.
2003, 125, 3192. (c) Agard, N. J.; Prescher, J. A.; Bertozzi,
C. R. J. Am. Chem. Soc. 2004, 126, 15046. (d) Krasinski,
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(19) Data for 10: IR (KBr): 2937, 1759, 1637, 1616, 1505, 1459,
1364, 1250, 1174, 1110, 1085, 834 cm–1. 1H NMR (DMSO-
d6, 300 MHz): d = 8.24 (1 H, s), 8.00 (1 H, d, J = 7.5 Hz),
7.79–7.69 (2 H, m), 7.32 (1 H, s), 7.28 (2 H, d, J = 8.4 Hz),
7.09 (1 H, d, J = 2.7 Hz), 6.81–6.74 (4 H, m), 6.65 (1 H, d,
J = 9.3 Hz), 6.59 (2 H, d, J = 1.2 Hz), 5.66 (1 H, s), 5.35 (1
H, dd, J = 12.6, 2.4 Hz), 5.22 (2 H, s), 4.36 (2 H, t, J = 6.6
Hz), 3.83 (2 H, s), 3.15 (1 H, dd, J = 17.1, 12.6 Hz), 2.69 (2
H, t, J = 7.2 Hz), 2.61 (1 H, dd, J = 17.1, 2.4 Hz), 2.40 (3 H,
s), 1.82 (2 H, t, J = 6.6 Hz), 1.56 (2 H, br m), 1.27 (4 H, br
m). 13C NMR (CDCl3, 75 MHz): d = 194.8, 173.4, 169.1,
162.6, 161.7, 160.2, 158.1, 152.8, 152.3, 142.6, 136.1,
130.6, 129.7, 129.5, 129.4, 128.7, 126.6, 125.1, 125.0,
124,5, 115.6, 113.4, 112.9, 111.9, 109.8, 102.7, 102.2,
100.0, 99.8, 96.6, 83.4, 78.5, 62.1, 55.7, 52.1, 49.8, 42.3,
40.4, 29.9, 26.1, 25.9, 25.3. HRMS: m/z calcd for
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Synlett 2006, No. 8, 1225–1229 © Thieme Stuttgart · New York