Chen et al.
enzyme
TABLE 3. Optical Properties and Bioactivities of the Labeled Compounds 27 and 28a b c
,
,
activity indexd e f
,
,
compd no.
λex1 (nm)
ε1 (cm-1 M-1
)
λem1 (nm)
Φ1
λex2 (nm)
ε2 (cm-1 M-1
)
λem2 (nm)
Φ2
27
28
453
481
39950
12339
537
526
0.09
0.07
479
30693
536
0.12
116.5
31.6
a All optical properties were measured in methanol. b Quantum yields were measured and calculated relative to quinine in 1 M H2SO4 as a standard
(excited at 478 nm). c Biological activities of the compounds were measured by ꢀ-galactosidase activity assays19 at the same cell density (OD600 of
0.4). d Naringenin derivatives (8 µM) were used in the experiments. e Enzyme activity index is the average results of at least 3 assays and is corrected
by the control values. f (()-Naringenin was used as the control with an enzyme activity index of 588.3.
SCHEME 6a
imaging27 clearly shows that naringenin-flu (27) permeated into
the Rhizobium cells, which retained livability and unceasing
random movements (see the Supporting Information for the
imaging picture). Since the size of Rhizobium cells is hundreds
of times smaller than that of mammalian cells, further imaging-
based studies on living Rhizobium cells are still technically
challenging with the current instrumental accuracy. Application
of naringenin-flu (27) to the FRET experiments and other
biological topics associated with nod-D protein and plant
nitrogen fixation is underway in this laboratory.
Conclusion
a Reagents and conditions: (a) BocNHCH2(CH2)8CH2NHCH3, HCHO,
EtOH, 81%; (b) TFA, CH2Cl2, then FITC, DIPEA, THF, 62%; (c) (HCHO)n,
N3CH2CH2(OCH2CH2)2NHCH3, ZnCl2, 65 °C, 76%; (d) 2, CuSO4 ·5H2O
(cat.), ascorbic acid, t-BuOH, H2O, rt, 84%.
A bioactive naringenin derivative, naringenin-flu (27), has
been acquired after global modification of the available positions
of natural product and further focused optimization. The mild
catalytic Huisgen 1,3-dipolar cycloaddition successfully served
as the common “Click” labeling tool in the last step. Various
methodologies developed for the site-specific modifications of
naringenin and for the facile syntheses of new naringenin
derivatives will be helpful in the biological applications of
naringenin and other flavonones. Naringenin-flu (27) was found
to acquire approximate 20% of the activity of naringenin, and
its entry into the living Rhizobium cells was unambiguously
observed in the corresponding fluorescent imaging experiment.
Both compounds 27 and 28 were found to exhibit satisfactory
optical properties according to the corresponding characteriza-
tions (Table 3). To our delight, naringenin-flu (27) acquires
approximately 20% of the activity of naringenin, though the
other thiocarbamide derivative 28 is inactive (Table 3). With
its satisfactory bioactivity and optical properties, naringenin-
flu (27) should be qualified for further biological experiments
in living Rhizobium cells.
Validation of the Use of a Racemic Probe. Though all
known reports in nod-D signalings and plant nitrogen-fixation
used the commercially available racemic naringenin (Sigma),17
it is still of importance to validate the rationality of employing
a racemic probe in further biological experiments. A straight-
forward way is to examine the difference of the biological effects
of (R)- and (S)-naringenin in regulating the nod-D signaling.
Both samples of (R)- and (S)-naringenin (>99% ee by HPLC)
were carefully prepared by chiral HPLC separation of the
racemic material.25 Their enzyme activities were then examined
under normal conditions.26 To our surprise, the results show
that (S)-naringenin, (R)-naringenin, and (()-naringenin exhibited
no observable difference in potency after 18 h of induction (see
the Supporting Information for details). To the best of our
knowledge, this is the first time such an unusual situation has
been experimentally demonstrated, though its cause remains
unknown. With these data, we thought the use of an enantiopure
probe of naringenin would be unnecessary in further applications.
Cell Permeability of Naringenin-Flu. Entry of naringenin-
flu (27) into Rhizobium cells was unambiguously confirmed by
visible evidence under a confocal microscope. Fluorescent
Experimental Section
Mannich Adduct 27a. To a solution of (()-naringenin (157 mg,
0.58 mmol) and paraformaldehyde (35 mg, 1.17 mmol) in ethanol
(5 mL) was added the secondary amine (MeNH-
(CH2CH2O)2CH2CH2N3) (130 mg, 0.69 mmol) at room temperature.
The mixture was then heated to 65 °C and stirred at this temperature
until the reactants were consumed. EtOAc (30 mL) and diluted
aqueous HCl (30 mL, pH 3) were added. The aqueous phase was
adjusted to pH 7 by adding solid NaHCO3, and then extracted with
EtOAc. The combined extracts were dried over anhydrous Na2SO4
and concentrated. The crude product was purified by flash column
chromatography (CH2Cl2/MeOH ) 50/1 to 15/1) to afford azide
27a (208 mg, 76%) as a white foam. IR (KBr) νmax 2878, 2110,
1679, 1638, 1519, 1459, 1344, 1303, 1170, 136, 1087, 836 cm-1
.
1H NMR (CDCl3, 300 MHz) δ 7.15 (2H, d, J ) 8.4 Hz), 6.67 (2H,
d, J ) 8.1 Hz), 5.59 (1H, s), 5.18 (1H, dd, J ) 12.6, 3.0 Hz), 3.61
(2H, s), 3.50-3.31 (8H, m), 3.15 (2H, t, J ) 4.8 Hz), 2.91 (1H,
dd, J ) 16.8, 12.6 Hz), 2.59 (2H, t, J ) 5.4 Hz), 2.48 (1H, dd, J
) 17.4, 3.0 Hz), 2.17 (3H, s). 13C NMR (CDCl3, 75 MHz) δ 196.0,
169.3, 162.5, 160.9, 157.7, 130.0, 128.1, 115.2, 101.2, 100.8, 95.5,
(27) Competent 8401/pKT230 cells (100 µL) were mixed with naringenin-
flu 27 (1 µL, 4 µM). The mixture was electroshocked at 1000 V for 4.5 ms with
a micropulser (wave shape PL5). The mixture was then incubated at 28 °C for
half an hour in darkness and centrifuged at 12 000 rpm for 10 min. The sediments
were washed with TEG buffer three times and re-solved in TEG buffer (50 µL).
Sample (5 µL) was dropped to the microplate and photos taken with a confocal
microscope. The excitation wavelength is 488 nm. The scale bar is 2 µm (for
details, see Figure S-1 in the Supporting Information).
(25) (a) Giorgio, E.; Parrinello, N.; Caccamese, S.; Rosini, C. Org. Biomol.
Chem. 2004, 2, 3602–3607. (b) Caccamese, S.; Caruso, C.; Parrinello, N.;
Savarino, A. J. Chromatogr. A 2005, 1076, 155–162.
(26) (a) Hu, H.-L.; Liu, S.-T.; Yang, Y.; Chang, W.-Z.; Hong, G.-F. Nucleic
Acids Res. 2000, 28, 2784–2793. (b) Sambrook, J.; Fritsch, E. F.; Maniatis, T.
Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, NY, 1989.
8284 J. Org. Chem. Vol. 73, No. 21, 2008