Synthesis of 3-hydroxyflavone fluorescent probes and study of their fluorescence properties

Article abstract of DOI:10.1016/j.cclet.2009.12.003

Direct measurement of dipole potential in biological membranes has been impossible and 3-hydroxyflavones (3HFs) have allowed detection of changes in dipole potential in biological systems. In the present study, sixteen derivatives of 3HF with aliphatic hy

Full text of DOI:10.1016/j.cclet.2009.12.003

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 529–532
www.elsevier.com/locate/cclet
Synthesis of 3-hydroxyflavone fluorescent probes and
study of their fluorescence properties
Xiao Dong Zhao a, Chang Jun Sun b, Qing Qiang Yao a, Wen Bao Li a,b,
*
a Institute of Materia Medica; Shandong Academy of Medical Sciences, Jinan 250062, China
b Sanlugen Pharma Tech, 18877 Jingshi Road, Jinan 250062, China
Received 31 August 2009
Abstract
Direct measurement of dipole potential in biological membranes has been impossible and 3-hydroxyflavones (3HFs) have
allowed detection of changes in dipole potential in biological systems. In the present study, sixteen derivatives of 3HF with aliphatic
hydrocarbon chains of different lengths at 40-position and 6-position were synthesized. The basic fluorescence properties of 3HFs
are maintained in all the probes in terms of strong blue shift in maximum fluorescence emission wavelength and >100 fold increase
in quantum yield in organic solvents and in dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) in comparison
to in aqueous Hepes buffer (15 mmol/L, pH 7.4). More importantly, the ability of the new compounds to report dipole potential
changes in biological systems are also maintained, since all the new probes showed spectrum properties that are similar to yet
different from that of F4N1, which potentially may allow more sensitive measurement of the dipole potential change in membranes.
# 2009 Wen Bao Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: 3-Hydroflavones (3HFs); Fluorescent probes; ESIPT; Dipole potential
3-Hydroxyflavones (3HFs) are dual-band fluorescent dyes due to an excited state intramolecular proton transfer
(ESIPT) reaction. The dual emission of 3HF is highly sensitive to the properties of the environment, such as polarity
and H-bond donor and acceptor ability. Therefore, they have been used as highly sensitive probes of their
microenvironments in biological systems [1,2]. It has been found that the introduction of an electron donor group to
the 40-position of the 3HF increased the sensitivity of fluorescence, and that the attachment of positively charged
ammonium group at 6-position of the 3HF dramatically changed the relative intensities of the two emission bands [3].
Accordingly, in the present study, two series, sixteen 3HFs derivatives including 14 new ones, were synthesized, and
the effect of aliphatic chains of different lengths at 40- and 6-positions of 3HFs on the fluorescence properties were
studied in organic solvents and in dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV).
1. Experimental
F2 and F4 two series, sixteen 3HFs derivatives including fourteen new ones, were synthesized according to the
synthetic route shown in Scheme 1.
* Corresponding author at: Sanlugen Pharma Tech, 18877 Jingshi Road, Jinan 250062, China.
E-mail address: wbli@sanlugen.com (W.B. Li).
1001-8417/$ – see front matter # 2009 Wen Bao Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2009.12.003

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X.D. Zhao et al. / Chinese Chemical Letters 21 (2010) 529–532
Scheme 1. The synthetic route of compounds 5: R1 = n-C2H5, or n-C4H9; R2 = CH3, n-C4H9, n-C6H13, n-C7H15, n-C8H17, n-C9H19, n-C10H21
n-C12H25. Condition and reagent: (a) H2O2, NaOH, EtOH, 0–60 8C; (b) 40% HBr, reflux 3 h; (c) N,N-dimethylalkylamine, EtOH, reflux 8 h.
,
Compound 1 and 2 were prepared according to the published methods [1,4]. Compound 3 with R1 = n-C2H5 or
n-C4H9 at 40-position, having the 3-hydroxyflavone core, were synthesized as the key intermediates using a
modification of Algar-Flynn-Ayamada reaction [5]. After the intermediates were bromided by 40% HBr to give
compound 4, the fluorescent probes (compound 5) could be obtained following the reaction with N,N-
dimethylalkylamine. The detailed synthesis procedure and all analytical data were reported as Ref. [6].
Stock solutions of all fluorescent probes were prepared by dissolving the probes in DMSO and then diluting with
Hepes buffer (15 mmol/L, pH 7.4) to obtain final concentration of 1 mmol/L (final DMSO concentration was <1% in
the DOPC SUV experiment). DOPC SUV was prepared according to the published article [7]. Fluorescence spectra
were recorded on a Hitachi F4500 spectrophotometer. Both N* and T* bands of the spectra were fitted by Gaussian
functions. UV–vis absorption spectra were performed on a Shimadzu UV2550 UV–vis spectrophotometer. All the
solvents were spectroscopic grade.
2. Results and discussion
F4N1 has been shown to be ultra sensitive to polarity and H-bonding capability of its microenvironment and has
been used to detect dipole potential changes in biological membranes [1]. When bound to liposomes or plasma
membranes of cells, it is located in the head group region of the phospholipid with the positively charged amine group
exposed to aqueous phase [1]. Fourteen new derivatives of 3HFs have been synthesized with the hope that they may
provide improved properties of F4N1 for detection of dipole potential in biological systems
Fig. 1 shows the fluorescence emission of F4N10 as an example of the new probes in Hepes buffer (15 mmol/L, pH
7.4), acetonitrile, dichloromethane, and when bound to DOPC SUV in Hepes buffer.
Fig. 1. Fluorescence emission of F4N10 at concentration 1 mmol/L in Hepes buffer (15 mmol/L, pH 7.4) (Black), in dichloromethane (Purple), in
acetonitrile (Green), and in DOPC SUV (Red) (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of the article.).

X.D. Zhao et al. / Chinese Chemical Letters 21 (2010) 529–532
531
Table 1
Spectroscopic properties of the probes in different organic solvents.
Compound 5
R1
Acetonitrile
Dichloromethane
R2
labs (nm) lN* (nm) lT* (nm) IN*/IT*
w
labs (nm) lN* (nm) lT* (nm) IN*/IT*
w
F2N1*
F2N4*
F2N6*
F2N7*
F2N8
n-C2H5 CH3
415
417
417
418
417
415
524
524
524
523
524
524
524
524
526
526
526
526
525
526
526
526
575
575
575
571
576
575
575
577
577
577
579
579
578
578
579
578
3.89
3.86
3.81
3.32
3.89
3.79
3.86
4.02
4.17
4.12
4.27
4.25
4.20
4.15
4.23
4.18
0.31 428
0.33 431
0.33 430
0.33 431
0.32 430
0.32 429
0.31 430
0.34 430
0.39 433
0.41 434
0.35 433
0.34 433
0.30 431
0.37 433
0.37 433
0.35 433
515
518
516
516
517
517
517
517
517
518
516
518
517
517
518
518
563
563
564
564
564
565
565
566
562
565
565
566
568
566
566
566
3.74
4.02
3.92
3.92
3.95
3.89
4.06
4.20
3.95
4.14
3.98
4.16
4.19
3.98
4.19
4.17
0.86
0.89
0.84
0.86
0.87
0.89
0.88
0.88
0.84
0.87
0.83
0.86
0.82
0.85
0.83
0.87
n-C2H5 n-C4H9
n-C2H5 n-C6H13
n-C2H5 n-C7H15
n-C2H5 n-C8H17
n-C2H5 n-C9H19
F2N9*
F2N10* n-C2H5 n-C10H21 418
F2N12* n-C2H5 n-C12H25 418
F4N1
n-C4H9 CH3
420
418
419
419
417
419
F4N4*
F4N6*
F4N7*
F4N8*
F4N9*
n-C4H9 n-C4H9
n-C4H9 n-C6H13
n-C4H9 n-C7H15
n-C4H9 n-C8H17
n-C4H9 n-C9H19
F4N10* n-C4H9 n-C10H21 419
F4N12* n-C4H9 n-C12H25 420
labs, position of absorption maxima; lN*and lT*, position of fluorescence maxima of N* and T* forms, lN*, lT* and IN*/IT* are obtained from
peakfit; w is the fluorescence quantum yield using F2N8 in acetonitrile as the reference (w = 0.32) [3]; excitation at 410 nm; *, new compound.
In Hepes buffer (15 mmol/L, pH 7.4), the fluorescence emission peak is found at 547 nm whose N* and I* bands are
not resolved well. Relative to that in Hepes buffer, the emission peak is blue shifted to 526 nm and 517 nm in
acetonitrile and dichloromethane, respectively; which also is accompanied by significantly increase in fluorescence
quantum yield. The blue shift in dichloromethane is more than in acetonitrile, due to higher apolarity of
dichloromethane. When bound to DOPC SUV, the emission peak also demonstrates blue shift with significantly
increased fluorescence quantum yield. More importantly, the new probe showed very pronounced increase in T* band
emission as indicated by the much decreased IN*/IT* value of 1.46 (Table 2).
Table 1 summarizes the spectroscopic properties of all the probes in acetonitrile and dichloromethane; Table 2
summarizes the spectroscopic properties of all probes in aqueous Hepes buffer (15 mmol/L, pH 7.4) and when bound
to DOPC SUV. All data show that they have maximum absorption and emission shifted to shorter wavelengths with
Table 2
Spectroscopic properties of the probes in Hepes buffer and DOPC SUV.
Probe
Hepes buffer (15 mmol/L, pH 7.4)
DOPC SUV
labs (nm)
lN* (nm)
lex (nm)
lN* (nm)
lT* (nm)
IN*/IT*
F2N1
F2N4
F2N6
F2N7
F2N8
F2N9
F2N10
F2N12
F4N1
F4N4
F4N6
F4N7
F4N8
F4N9
F4N10
F4N12
429
430
431
434
436
433
432
441
436
437
440
443
448
450
449
452
550
549
550
551
551
553
552
552
554
553
556
552
554
553
547
547
423
423
424
424
424
424
425
425
426
427
427
427
428
428
427
428
489
494
499
499
501
502
501
504
492
496
499
501
501
502
502
503
548
547
552
549
554
555
553
558
546
553
559
561
562
562
563
563
0.89
0.68
0.84
0.96
0.96
0.99
0.93
1.14
1.12
1.24
1.4
1.40
1.40
1.44
1.46
1.46
lex, position of excited spectroscopic maxima recorded at 500 nm; lN* and lT*, position of fluorescence maxima of N* and T* forms; excitation
wavelength 400 nm; lN*, lT* and IN*/IT* are obtained from peakfit.

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X.D. Zhao et al. / Chinese Chemical Letters 21 (2010) 529–532
significantly increased fluorescence quantum yield. Titration of all probes with DOPC SUV has indicated a drastic
increase in quantum yields (date not shown).
Increase of the length of aliphatic chain at 6-position of 3HFs from one carbon to 12 carbons results in a gradual red
shift of both excitation and emission maxima in both F2 and F4 series, which is accompanied by increased intensity
ratio of N* and T* band, IN*/IT* (Table 2). This suggests that as the aliphatic chain length increases, the force to pull the
3HF moiety into the membranes from the positively charged side is increased; since the membrane is a strong barrier
for cations to enter, the 3HF moiety has to adopt a different conformation in e.g. F4N12 than in F4N1 and the net result
is that the 3HF moiety is buried shallower in the phospholipid head group region. This should allow 3HF moiety in for
example F4N12 to detect changes of dipole potential in the membrane at a different position and depth in the
membrane, and this possibility is being explored.
Acknowledgment
We gratefully thank Dr. Bing Yan for the use his fluorescence and UV–vis spectrophotometers.
References
[1] A.S. Klymchenko, G. Duportail, T. Ozturk, et al. Chem. Biol. 9 (2002) 1199.
[2] A.S. Klymchenko, G. Duportail, Y. Mely, et al. Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 11219.
[3] A.S. Klymchenko, A.P. Demchenko, J. Am. Chem. Soc. 124 (2002) 12372.
[4] A. Ulman, C.S. Willand, W. Kohler, et al. J. Am. Chem. Soc. 112 (1990) 7083.
[5] M.A. Smith, R.M. Neumann, R.A. Webb, J. Heterocycl. Chem. 5 (1968) 425.
[6] Synthesis procedures: Compound 3 was prepared using a modification of Algar-Flynn-Ayamada reaction: to the solution of compound 1 (35 mmol)
and compound 2 (35 mmol) (R1 = n-C2H5, or n-C4H9) in EtOH (40 mL), 15 mL of aqueous solution of NaOH (10 g, 250 mmol) was added, the
mixture was stirred at r.t. for overnight. To the resultant dark red solution, 10 mL of NaOH aqueous solution (2.5 g, 62.5 mmol) was added; the
temperaturewasloweredtobelow15 8C, 18 mLof30%H2O2 solutionwasadded. Thenthetemperaturewasraisedto40–60 8Candstirredfor0.5 h.
After the reaction, pH was adjusted to 7 with diluted H2SO4 and extracted with ethyl acetate. The extracted compound 3 was concentrated with
vacuum, and purified with silica gel to yield a bright yellow solid compound 3. Compound 3 in 40% HBr solution (10 mL) was refluxed for 3 h, then
the mixture was neutralized with NaHCO3 and the precipitatewas filtered to give compound 4, which was used directly without further purification.
Compound 5 were prepared as following: compound 4 (0.60 mmol) and N,N-dimethylalkylamine (0.70 mmol) was dissolved in ethanol (10 mL)
and refluxed 8 h. After removal of solvents, the solid product was recrystallized with CH2Cl2 and ether to get compound 5. Using the similar
procedures, 16 derivatives of 3HFs were prepared and reported in Table 1. The melting points were determined on a RY-IG melting point apparatus
andwereuncorrected. 1HNMRspectrawererecordedonaVarian600 MHzspectrometerusingtetramethylsilaneasaninternalstandard. HPLC–MS
were recorded on Agilent 1100 Series LC/MSD. Selected analytical data for probes (compounds 5): F2N4: yield: 69%, m.p. 180–181 8C, 1H NMR
(600 MHz,CDCl3):d1.00(t, 3H, J = 7.2 Hz),1.23(t, 6H, J = 7.2 Hz),1.45(m, 2H),1.85(m, 2H),3.34(s, 6H),3.46(m, 4H),3.62(t, 2H, J = 8.4 Hz),
5.25 (s, 2H), 6.74 (d, 2H, J = 9 Hz), 6.92 (br, 1H), 7.58 (d, 1H, J = 9 Hz), 8.11 (d, 2H, J = 9 Hz), 8.22 (d, 1H, J = 1.2 Hz), 8.30 (dd, 1H, J = 8.4 Hz,
J = 1.8 Hz), LC-MS: m/z 423 [M-Brꢀ]+, (C26H35BrN2O3, 502). F2N6: yield: 71%, m.p. 145–147 8C, 1H NMR (600 MHz, CDCl3): d 0.87 (t, 3H,
J = 7.2 Hz), 1.23 (t, 6H, J = 7.2 Hz), 1.28–1.38 (m, 6H), 1.84 (m, 2H), 3.33 (s, 6H), 3.44 (m, 4H), 3.59 (t, 2H, J = 9 Hz), 5.25 (s, 2H), 6.73 (d, 2H,
J = 9.6 Hz), 6.97 (br, 1H), 7.55 (d, 1H, J = 9 Hz), 8.10 (d, 2H, J = 9 Hz), 8.23 (s, 1H), 8.28 (dd, 1H, J = 9 Hz, J = 1.8 Hz), LC-MS: m/z 451 [M-
Brꢀ]+, (C28H39BrN2O3, 530). F2N8: yield: 63%, m.p. 154–156 8C, 1H NMR (600 MHz, CDCl3): d 0.86 (t, 3H, J = 6.9 Hz), 1.22–1.83 (m, 18H),
3.33 (s, 6H), 3.45 (q, 4H, J = 7.2 Hz), 3.59 (t, 2H, J = 8.4 Hz), 5.26 (s, 2H), 6.74 (d, 2H, J = 9 Hz), 6.9 (br, 1H), 7.60 (d, 1H, J = 9 Hz), 8.12 (d, 2H,
J = 9 Hz), 8.22 (d, 1H, J = 1.8 Hz), 8.33 (dd, 1H, J = 9 Hz, J = 1.8 Hz). F2N10: yield: 74%, m.p. 169–171 8C, 1H NMR (600 MHz, CDCl3) d: 0.86
(t, 3H, J = 7.2 Hz), 1.22–1.84 (m, 22H), 3.33 (s, 6H), 3.45 (q, 4H, J = 7.2 Hz), 3.60 (t, 2H, J = 8.4 Hz), 5.25 (s, 2H), 6.73 (d, 2H, J = 9 Hz), 6.93 (br,
1H), 7.58 (d, 1H, J = 9 Hz), 8.11 (d, 2H, J = 9 Hz), 8.22 (d, 1H, J = 1.8 Hz), 8.31 (dd, 1H, J = 9 Hz, J = 1.8 Hz). F2N12: yield: 76%, m.p. 157–
159 8C, 1H NMR (600 MHz, CDCl3): d 0.87 (t, 3H, J = 6.9 Hz), 1.22–1.82 (m, 28H), 3.32 (s, 6H), 3.45 (q, 4H, J = 7 Hz), 3.58 (t, 2H, J = 8.1 Hz),
5.24 (s, 2H), 6.74 (d, 2H, J = 9 Hz), 7.58 (d, 1H, J = 8.4 Hz), 8.11 (d, 2H, J = 9 Hz), 8.22 (s, 1H), 8.32 (d, 2H, J = 7.8 Hz), LC-MS: m/z 535 [M-
Brꢀ]+, (C34H51BrN2O3, 614). F4N1: yield:58%, 1H NMR (600 MHz, DMSO-d6): d 0.92-0.95 (t, 6H, J = 7.2 Hz), 1.33–1.37 (m, 4H), 1.52–1.57 (m,
4H), 3.07(s, 9H), 3.35–3.38 (t, 4H, J = 7.8 Hz), 4.70(s, 2H), 6.79–6.80(d, 2H, J = 9 Hz), 7.86(t, 2H), 8.10–8.12 (d, 2H, J = 9 Hz), 8.29(s, 1H), 9.35
(s, 1H), MS: m/z437 [M-Brꢀ]+, (C33H49BrN2O3, 516). F4N6: yield: 59%, m.p. 164–166 8C, 1H NMR (600 MHz, CDCl3): d 0.87 (t, 3H, J = 6.9 Hz),
0.98(t, 6H, J = 7.2 Hz),1.29–1.44 (m, 10H), 1.61(m, 4H), 1.83(m,2H),3.34(s, 6H), 3.35(m, 4H), 3.58(t, 2H, J = 8.4 Hz), 5.23(s, 2H), 6.71(d, 2H,
J = 9 Hz), 6.91 (br, 1H), 7.58 (d, 1H, J = 9 Hz), 8.11 (d, 2H, J = 9 Hz), 8.23 (s, 1H), 8.31 (d, 1H, J = 8.4 Hz), LC-MS: m/z 507 [M-Brꢀ]+,
(C32H47BrN2O3, 586). F4N10: yield:69%, m.p. 157–159 8C, 1H NMR (600 MHz, CDCl3):d 0.87(t, 3H, J = 6.9 Hz), 0.99(t, 6H, J = 7.2 Hz), 1.23–
1.84 (m, 24H), 3.36 (m, 10H), 3.60 (t, 2H, J = 8.4 Hz), 5.26 (s, 2H), 6.71 (d, 2H, J = 9 Hz), 6.88 (br, 1H), 7.60 (d, 1H, J = 8.4 Hz), 8.12 (d, 2H,
J = 9 Hz), 8.22 (s, 1H), 8.34 (d, 1H, J = 8.4 Hz), LC-MS: m/z 563[M-Brꢀ]+, (C36H55BrN2O3, 642). F4N12: yield: 72%, m.p. 166–168 8C, 1H NMR
(600 MHz, CDCl3): d 0.87 (t, 3H, J = 7.2 Hz), 0.99 (t, 6H, J = 7.2 Hz), 1.23–1.42 (m, 22H), 1.62 (m, 4H), 1.83 (m, 2H), 3.34–3.37 (m, 10H), 3.60 (t,
2H, J = 8.4 Hz), 5.26 (s, 2H), 6.73 (d, 2H, J = 9 Hz), 6.83 (s, 1H), 7.62 (d, 1H, J = 8.4 Hz), 8.13 (d, 2H, J = 8.4 Hz), 8.20 (s, 1H), 8.38 (dd, 1H,
J = 8.4 Hz, J = 1.8 Hz). Analytical data for other probes, F2N1, F2N7, F2N9, F4N4, F4N7, F4N8 and F4N9 are also available by request.
[7] J.M. Leenhouts, P.W.J. Van den Wijngaard, A.I.P.M. De Kruijff, et al. FEBS Lett. 370 (1995) 189.