Sensors and Actuators B 102 (2004) 320–324

The fluorescence studies of interaction between 4-(n-2
-glucosyl)

butyramidyl triphenyl phosphonium chloride and DNA
Gong-Wu Song∗ , Zhao-Xia Cai, Yu He, Zhao-Wen Lou
Faculty of Chemistry and Material Science, Hubei University, Wuhan 430062, PR China
Received 22 October 2003; received in revised form 11 April 2004; accepted 26 April 2004

Available online 1 June 2004

Abstract

A new compound 4-(n-2
-glucosyl) butyramidyl triphenyl phosphonium chloride (4-GBTPC) was synthesized, and its interactions with
nucleic acid were studied by fluorescence method. The results of the experiment indicated that DNA can obviously decrease the fluorescence
intensity of 4-GBTPC by the static quenching style. DNA could be determined by using of the fluorescence quenching action of DNA on
4-GBTPC. The linear range was 8.9 × 10−6 to 1.16 × 10−4 mol L−1 with the detection limit of 9.1 × 10−7 mol L−1 . Effects of interference
substances and optimization of procedure were investigated. Three synthetic samples were determined with satisfaction.
© 2004 Elsevier B.V. All rights reserved.

Keywords: 4-(n-2
-Glucosyl) butyramidyl triphenyl phosphonium chloride; Fluorescence; Static quenching style; DNA

1. Introduction dosage of d-glucosamine hydrochloride has no harm for

host tissue.
Cancer is one of the diseases most difficult to be cured. So it is necessary to synthesize and characterize more
It is very important to find most efficiency new anticancer phosphides and study the relations between their structure
drugs [1–3]. Much money and time have been taken to and activity. Our research group synthesized a new com-
choose new anticancer drugs in clinical treatment meth- pound 4-(n-2
-glucosyl) butyramidyl triphenyl phosphonium
ods. So it is necessary to find a simpler and more efficient chloride. The molecular structure contains glucosamine and
methods of discovering new drugs. All the anticancer drugs phosphonium groups, as shown in Fig. 1. We can presume
so far found interact with DNA molecules, so that binding that 4-GBTPC has anticancer activity because of the two
studies of small molecules with DNA are important in the functional groups. The study of interaction between them
design of new and efficient drugs targeted to DNA. will be beneficial for medical science and pharmacokinetics.
Ammonium salts as disinfectants have been employed Many methods such as NMR [13], HPLC [14] have been
widely. It is recently shown that phosphonium salts are employed in the study of interactions between phosphide
charged particles as in the case of ammonium salts and have with macromolecules. As one of the modern instrumental
an affinity to cells, and the interference of cell metabolism analytical methods the fluorescence analysis is developing
of phosphine is stronger because of participation of phos- fast in recent years and playing an important role in the
phorus. There has also been certain interest in the studies of study on bioscience due to its high sensitivity and selec-
phosphide as antibiotics and its sterilizing effect [4]. Studies tivity. DNA can quench the fluorescence intensity of many
showed that some triphenylphosphonium salts have anti- pharmic molecules, especially quinolone antibacterial agents
cancer activity to mice [5–8]. d-Glucosamine hydrochloride [15–17].
has a good antibacterial and antiphlogistic function [9]. It This paper focuses on the application of fluorescence anal-
is an important material for synthesis of anticancer drugs ysis in the study on the interaction between medicine and
[10–12] and is widely used in medicine preparations. Large deoxyribonucleic acid, and suggests that DNA can obvi-
ously decrease the fluorescence intensity of 4-GBTPC by
the static quenching style. The calibration linearity for DNA
∗
extends up to 4.8 × 10−5 mol L−1 as a lower determination
Corresponding author. Tel.: +86-2750865305.
E-mail addresses: songgw@hubu.edu.cn (G.-W. Song), limit. Under the experimental conditions studied, there is lit-
hubucaicai@yahoo.com.cn (Z.-X. Cai). tle interference form proteins, nucleosides and most metal

0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2004.04.089
 G.-W. Song et al. / Sensors and Actuators B 102 (2004) 320–324 321

lar of 1 M KOH, mixed and stirred. Then an equimolar

4-butanoyl chloride was added to the above solution in
ice-bath and distillated under reduced pressure. After filtra-
tion the obtained crystal was dissolved in a small amount
of water and annealed in anhydrous ethanol, followed by
filtration and dryness in vacuum. The above product mixed
with an equimolar amount of triphenyl phosphonium in
methanamide, and anhydrous enthol as dispersant agent at
90 ◦ C, followed by stirring for 8 h. Then, the products were
Fig. 1. Structure of 4-GBTPC.
purified with a short column chromatography on silica gel
using a solution of ethyl acetate and acetaldehyde, followed
ions. Interference by some metal ions, surfactants and salts
by filtration and dryness. Yield was 8%.
can be minimized by dilution. Binding constant and num-
ber between them can be determined. The work also sheds
light on the interaction mechanism of phosphide with macro- 2.4. Procedures
molecules.
In a dry 25 ml volumetric flask are added 0.2 ml of the
4-GBTPC solutions, 1.0 ml of Tris–HCl solution, and an
2. Experimental appropriate volume of nucleic acid. The mixture was di-
luted to 12.5 ml with doubly distilled water and vortexes.
2.1. Apparatus Five minutes later, the fluorescence spectra were measured
with the following settings of spectrofluorometer: emission
The fluorescence spectra were measured with a Shimadzu wavelength (λem ) of 275 nm; excitation wavelength (λex )
RF-540 spectrofluorometer with a 1 cm ×1 cm cross-section of 590 nm, and the excitation and emission slits are 10 nm.
quartz cell (Kyoto, Japan). The absorption measurements Then all the absorption and fluorescence spectra were ob-
were performed on a Perkin-Elmer lambda 17 UV/VIS spec- tained against the blank treated in the same way without
trophotometer (P-E Co., USA). A WH-2 vortex mixer (Huxi nucleic acid.
Instrumental Co., Shanghai, China) was used to blend the
solution, and a PHB-4 pH meter is used to measure the pH
of the solution. A super thermostat circulating water bath 3. Results and discussion
was used for maintaining the difference temperatures of the
systems for the fluorescence quenching experiments. 3.1. Studies between 4-GBTPC and DNA by spectral
method
2.2. Reagents
Typical emission spectra of 4-GBTPC with and without
All reagents for synthesis were of analytical reagent DNA were shown in Fig. 2. The existence of DNA can
grade unless otherwise stated. Doubly distilled water is quench the intensity of fluorescence of 4-GBTPC at the
used throughout the work. excitation wavelength of 355 and 590 nm. This quenching
The working concentration of 4-GBTPC solution was
2.0 × 10−4 mol L−1 . The stock solution of DNA was pre-
pared by dissolving commercially purchased calf thymus
DNA (Baitai Biochemical Co., Chinese Academy of Sci-
ences) in doubly distilled water at 0–4 ◦ C. Twenty-four hours
or more were needed for dissolving DNA with occasionally
gentle shaking. The concentrations of stoke solutions of nu-
cleic acids were determined by the absorbance at 260.0 nm.
The working concentration of the nucleic acid solution was
4.4 × 10−4 mol L−1 , which was prepared by diluting the
stock solution with deionized water. Tris (hydroxymethyl)
aminomethane–HCl solution is used to control the acidity.
0.1 mol L−1 NaCl was used to adjust the ionic strength of
the aqueous solutions.

2.3. Synthesis of 4-GBTPC

Fig. 2. Fluorescence spectra of 4-GBTPC–DNA system. C4-GBTPC =
Glucosamine chloride was dissolved in a small amount 6.4 × 10−6 mol L−1 , CDNA = 2.96 × 10−5 mol L−1 . 1. 4-GBTPC; 2.
of doubly distilled water, and reacted with an equimo- 4-GBTPC + DNA.
322 G.-W. Song et al. / Sensors and Actuators B 102 (2004) 320–324

spectrum indicated that 4-GBTPC could interact with DNA. 1.2

The decreased fluorescence intensity of 4-GBTPC was pro- 1
portional to the concentration of nucleic acids. Based on this 0.8

( F0/F)-1
decrease, the amount of nucleic acid could be determined. 0.6
0.4
Fig. 3 shows the absorption spectra of the 4-GBTPC–DNA
0.2
systems. There are broad absorption peaks at the wave-
0
length 225 nm. With increasing the DNA concentration, the 0 20 40 60 80 100 120
-6
absorption at 225 nm increased. At the same time, a new [DNA](10 mol/L)
peak at 263 nm appeared. These phenomena also indicated Fig. 4. Fluorescence quenching Stern–Volmer plots of 4-GBTPC with in-
the conformation of 4-GBTPC and DNA. creasing concentration of DNA. C4-GBTPC = 6.4 × 10−5 mol L−1 . Round-
ness: 35 ◦ C; triangle 45 ◦ C.
3.2. Effect of temperatures on the system
ture, K35 ◦ C = 10 400 L mol−1 and K45 ◦ C = 8700 L mol−1 .
The above result may suggest the formation of a binary The static quenching is attributable to the formation of a
complex of 4-GBTPC–DNA. In order to affirm this conclu- non-fluorescence ground state complex between 4-GBTPC
sion, effect of temperature of the system was studied. and DNA.
The efficiency of quenching of a fluorophore species by
a quencher species follows the Stern–Volmer relationship:
3.3. The composition of the binary complex
F0
= 1 + Ksv [Q] (1)
F The composition of the binary complex can be deduced
from the following formula [18]:
where F0 and F are the fluorescence intensities in the ab-
sence and presence of the quencher, respectively. Either the M + nL = MLn (2)
collisional quenching of fluorescence or the static quench-

F0 − F
ing of fluorescence can be described by the Stern–Volmer log = logKa + n log[M] (3)
equation. [Q] is the concentration of the quencher. Ksv is F
the Stern–Volmer quenching constant. If a system obeys the where M is the quencher, and L the pharmaceutical molecule
Stern–Volmer equation, a plot of F0 /F versus [Q] will give with a fluorophore, MLn the binary complex whose resultant
a straight line with a slope of Ksv and a y-axis intercept. For constant is Ka, F0 the fluorescence of the total amount of the
dynamic quenching, Ksv increases with increasing the sol- pharmaceutical molecule (bound and unbound), and F the
vent temperature. And for static quenching, Ksv decreases fluorescence of unbound pharmaceutical molecule. A plot
with increasing the temperature. of log[(F0 − F)/F ] versus log[M] will give a straight line
Fig. 4 shows the Stern–Volmer plot of the 4-GBTPC–DNA with a slope of n and a y-axis intercept log Ka.
systems. In Fig. 4, the term, F0 /F, linearly increase with Fig. 5 is obtained by keeping the 4-GBTPC concentration
increased concentration of quencher. The coefficients are (2.54 × 105 mol L−1 ) constant and changing the concentra-
0.997 (35 ◦ C) and 0.9934 (45 ◦ C). At the same time, the tion of DNA. The data are well fitted to Eq. (3) and the
quenching efficiency of 4-GBTPC fluorescence by DNA slope was 1.4. The coefficient was 0.9952. The result indi-
underwent an intense decrease with increasing the tempera- cates that the fluoroquibolone antibiotic can form a stable
1:1.4 complex with DNA.

3.4. Effect of ionic strength

The effect of ionic strength was controlled by 0.1 mol L−1

NaCl and MgCl2 solutions. The experimental result is shown

0.5
0
Log[(F 0-F)/F]

-0.5
-1
-1.5
-2
-5.5 -5 -4.5 -4 -3.5
Log [DNA]
Fig. 3. UV spectra of 4-GBTPC–DNA system. C4-GBTPC =
6.4 × 10−6 mol L−1 , CDNA = 2.96 × 10−5 mol L−1 . 1. 4-GBTPC + DNA; Fig. 5. Estimation of composition of the 4-GBTPC–DNA complex.
2. 4-GBTPC; 3. DNA. C4-GBTPC : 3.2 × 10−5 mol L−1 .
 G.-W. Song et al. / Sensors and Actuators B 102 (2004) 320–324 323

Table 1
Interferences of foreign substances
Foreign substances Concentration (mg L−1 ) Change of IFI (%)

Zn2+ 160 4.87

Cu2+ 80 −0.7
K+ 160 −4.3
Ba2+ 160 2.79
Ni2+ 1.28 1.64
Mn2+ 25.6 4.62
As5+ 6.4 1.36
Cr3+ 80 4.62
Ag+ 1.38 0.005
Pb2+ 2.56 3.38
Sn4+ 1.28 0.528
Hg2+ 6.4 4.63
Br− 0.0819 2.12
l-Cysteine 0.384 0.877
BSA 1.6×10−6 2.23

Fig. 6. The effect of ionic strength on the 4-GBTPC–DNA sys-

tem. C4-GBTPC : 3.2 × 10−5 mol L−1 , CDNA : 2.96 × 10−5 mol L−1 1. 3.6. Tolerance of foreign substances
4-GBTPC + DNA + NaCl; 2. 4-GBTPC + DNA + MgCl2 .
The influence of foreign coexisting substances that were
exited in biological fluids, such as metal ions, protein and
in Fig. 6. With an increase in the concentration of metal amino acid were tested. The results are presented in Table 1.
ions, the fluorescence intensity of the 4-GBTPC–DNA sys- Among the metal ions tested, Zn2+ , K+ , Ca2+ and Mn2+
tem increased slightly. The maximum fluorescence intensi- can be allowed with relatively higher concentration levels.
ties were obtained at 0.17 and 0.14 mol L−1 for Na+ and It has been proved that they tend to bind exclusively to
Mg2+ , respectively. Then, that of the blank solution changed the phosphate groups of DNA molecules, stabilizing the
little. Therefore, the ionic strength has some effect on the Watson–Crick double-stranded helix [19]. In contrast, those
system, and we can conclude that Na+ has a good effect on ions, such as Sn4+ and Ag+ , which appear to bind exclu-
the system. Thus 2 ml of 0.1 mol L−1 NaCl is selected to sively with the base moiety of DNA to form internal chelates,
control the ionic strength. interstrand complexes or cross-links [19], can be allowed
with relatively low concentration. Although the ions, BSA
3.5. Effect of denatured DNA and natural DNA and l-cysteine can be tolerated at relatively low levels, their
quantities in samples of biological fluids diluted for analysis
The behaviors of native DNA and denatured DNA are usually below the amounts tolerated under the experi-
were compared. Double-strand DNA was converted into mental conditions.
single-strand DNA with the opening of its double helix by
incubation at 100 ◦ C for 30 min and immediately cooling in 3.7. Calibration curve
ice water. The experiments showed that natural DNA could
quench the fluorescence of 4-GBTPC linearly. But denatured A calibration curve was obtained according to the above
DNA cannot quench the fluorescence intensity of 4-GBTPC standard procedure. A linear relationship existed between
at all. The natural DNA split into two string-like soft the fluorescence intensity and the concentration of nucleic
polynucleotide chains from the original rigid double-helix acid when the concentration of nucleic acid was in the range
structure, which caused the difference in the fluorescence of 8.9 × 10−6 to 1.16 × 10−4 mol L−1 . The linear regression
quenching. The main reason may be associated with the equation was I = 79.411 − 0.335C (DNA), r = 0.9943.
intercalation of 4-GBTPC into native DNA base pairs. The detection limit (3σ) was 9.1 × 10−7 mol L−1 .

Table 2
Determination results of synthetic samples
Sample Concentration of DNA (×10−5 mol L−1 ) Foreign substances Found (×10−5 mol L−1 ) Recovery (%)

CtDNA 4.32 Pb2+ , Sn4+ , Mn2+ 4.35 100.7

CtDNA 8.64 Cr3+ , l-cysteine 8.51 98.49
CtDNA 12.96 Zn2+ , K+ , Ca2+ 13.1 101.08

Concentrations: CPb2+ = 0.02 mg L−1 ; CSn4+ = 0.02 mg L−1 ; CMn2+ = 2.4 × 10−6 mg L−1 ; CCr3+ = 0.01 mg L−1 ; Cl-cysteine = 0.41 mg L−1 ; CZn2+ =
0.04 mg L−1 ; CK+ = 0.01 mg L−1 ; CCa2+ = 0.01 mg L−1 .
324 G.-W. Song et al. / Sensors and Actuators B 102 (2004) 320–324

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fluorescence intensity of 4-GBTPC by the static quenching
style. This method has the following merits: high sensitiv-
ity, mild reaction condition, good reproducibility, a lower Biographies
detection limit and relatively few interfering substances.
The work also sheds light on mechanism of interaction Gongwu Song received his BS in analytical chemistry in Hubei University
between phosphate and macromolecules. from 1980 to 1984. Currently, he is a professor and work in Faculty of
Chemistry and Material Science, Hubei University (Wuhan, China) from
1984 to 2004. His research interests include fluorescence spectrometry
and its combination with FI, UV spectroscopy as well as HPLC for nu-
Acknowledgements cleic acid and protein biochemical analysis.

The authors thank the China Association for financial Zhaoxia Cai received her BS in Faculty of Chemistry and Material Sci-
support. ence, Hubei University from 1997 to 2001 (Wuhan, China). Her research
interests include fluorescence spectrometry and UV spectroscopy for nu-
cleic acid analysis.
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