The interaction of 6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b] pyridine-5-carbonitrile with two kinds of snake venom: A fluorescence quenching study

Lian Shuqin

    Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule. Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and quencher, or static, resulting from the formation of a ground-state complex between the fluorophore and quencher [1]. In both cases, molecular contact is required between the fluorophore and the quencher for fluorescence quenching to occur [2]. Application of the fluorescence quenching technique can also reveal the accessibility of the fluorophores to quenchers.
    The combination of drug and protein is an importantly pharmic and kinetic character. Studying the fluorescence quenching of pharmaceutical molecular to proteins, we can get many relative information about proteins molecular, so the study is widely much accounted of.
    There have been no studies on fluorescence quenching of snake venom induced by drugs or other small molecules. In this paper, we synthesized 6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5- carbonitrile (Fig. 1) and their interactions with the two kinds of snake venom were subsequently studied. And the interactions of I with cobra venom and viper venom were researched respectively in detail. The quenching constants KSV, binding constants K and binding sites n based on the fluorescence quenching were calculated.

Fig. 1 Molecular structure
R = H, 2-OH, 4-NO2, 4-CN, 4-Br, 4-Cl, 4-CH3, 2-Cl, 4-OCH3, 3-NO2, 4-N(CH3)2

    2.1 Reagent and tnstrumentation
    Cobra venom and viper venom were purchased from the of in Wuyi Mountain Snake Research Institute (Fujian, China). 6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile were synthesized from aromatic aldehyde, malononitrile and 1-phenyl-3-methyl-1H-pyrazol-5-amine (recrystallized 2 times with 95% ethanol, normalized purity (HPLC) 99.50 – 99.86%). 0.9% (w/v) NaCl aqueous solution was used to keep biologically ionic strength. The Tris (hydroxymethylaminomethane) buffer (> 99.5%) and other agents were of analytical purity. Redistilled water was used throughout.
    An F-4500 spectrophotometer (Hitachi, Japan) was used and the cell dimension is 1×1×4 cm3, the slit width was 3 nm. The pH values were measured with a Aiwang (Shanghai, China) pH meter.
    2.2 Experimental process

    The following reagents were added to a 10 mL tube in the order indicated: 1.0 mL of venom (1.0 mg/mL), adequate I (100 ug/mL) and 2.0 mL of Tris-HCl buffer solution (0.05 mol/L, pH = 7.6). After adding 0.9% NaCl aqueous solution to the tube, the absorption spectra and fluorescence spectra were measured at room temperature. Fluorescence measurements were taken at excitation and emission wavelengths of 282 and 300~500 nm, respectively, with a resolution of 3 nm.
    3.1 Fluorescence spectra

    Fig. 2 were fluorescence spectra of which cobra venom and viper venom respectively mixed (v: v = 1: 1).


Fig. 2 The emission spectrum of cobra venom (a) and viper venom (b) at room temperature and 282 nm.
1. cobra venom or viper venom (0.1 mg/mL)
2. I (10 ug/mL)
3. cobra venom-I or viper venom-I (0.1 mg/mL) + I (10 ug/mL)

From Fig. 2, we can observe an obvious decrease in the relative fluorescence intensity (RFI) of cobra venom and viper venom, but the intensity of I was not foundamental changed. That was to say there existed mutual action and stabilizing complex which is on ground state and no fluorescence was formed. The fluorescence of two kinds of venom may be due to the tryptophane in molecular chain [3]. So the content of tryptophane in corbric venom may be more than that one in viper venom according to Fig. 2.
3.2 The binding properties and quenching mechanism
The fluorescence quenching of cobra venom and viper venom with varying concentrations of I is shown in Fig. 3.



Fig. 3 a. Fluorescence spectra of 0.1 mg/mL cobra venom at l ex = 282 nm showing the quenching effect of increasing concentrations of I (0, 3, 5, 7, 10, 12, 15, 18, 20 mg/mL). Spectra were recorded at pH 7.6.
b. Fluorescence spectra of 0.1 mg/mL viper venom at l ex = 282 nm showing the quenching effect of increasing concentrations of I (0, 3, 5, 7, 10, 12, 15, 18 mg/mL). Spectra were recorded at pH 7.6.

Fluorescence quenching is described by the well-known Stern–Volmer equation:
where F0 and F denote the steady-state fluorescence intensities in the absence and in the presence of quencher I, respectively, KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of the quencher. Hence, the equation was applied to determine KSV by linear regression of a plot of F0/F against [Q].
The fluorescence of two kinds of venom were quenched by I. Respectively, the quenching Stern-Volmer figures of cobra venom and viper venom with varying concentrations of I are shown in Fig. 4. It showed that both of curves have linear relation (r= 0.992, 0.995). The constants KSV can be caculated (Table 1).

Fig. 4 The Stern-Volmer plot of cobra venom and viper venom quenching spectrum
1. cobra venom-I 2. viper venom-I

Table 1 quenching constants KSV、binding constants K and binding sites n

cobra venom-I 1.73 0.89 0.38
viper venom-I 2.32 1.05 0.44

To dynamic quenching style, Ksv= Kd= Kqt 0. To biomacromolecule, t 0= 108 s, qsv,max= 2.0×1010 L× mol– 1 × s-1 [5]. If the quenching phenomenon of venom and I were dynamic, rate constant kq should far more than 2.0×1010 L× mol-1× s-1. The result in the paper is opposite, so the quenching reason was that two styles formed new compounds and they were static quenching proceedings. When considering the effect of I on the fluorescence spectra of two kinds of venom, there was no apparent l em shift. This suggests no other change in the immediate environment of the fluorophores unless I is close to the fluorophores to occur the quenching effect. This means that the molecular conformation of the two kinds of venom is affected.
So the quenching reason was that a new steady complex which has no fluorescence was formed and it was a static quenching proceeding with the equation:
Log (F0-F)/F = Log K + n Log [Q]
The binding constants K and binding sites n could be calculated from Fig. 4 (r = 0.991, 0.992), and the calculated results (Table 1) indicated that it had strong binding between I and two kinds of snake venom.
3.3 Effect of substituted group of different aromatic aldehyde
Other 11 aromatic aldehydes (R=-H, 4-NO2, 4-CN, 4-Br, 4-Cl, 2-OH, 4-CH3, 2-Cl, 4-OCH3, 3-NO2, 4-N(CH3)2) were also researched. The result indicated that only I (R=2-OH) can do so. We supposed that this activity was mainly come from –OH which can bind with two kinds of venom through hydrogen bond.

    It was demonstrated the two proceedings are static. According to the maximum excitation and the maximum emission, we can infer that tryptophan may be the component of cobra venom and viper venom from Fujian Province and the content of tryptophan in viper venom is less than in cobra venom.
    I can interact with cobra venom and viper venom. Whether I can reduce the toxicity of snake that needs to be further investigated in future work.

ACKNOWLEDGEMENTS   We are grateful to the foundation of the “Natural Science Research Project of University in Jiangsu Province” (No. JH03-038) for financial support.

, Wu Yunming, Yin Xiaoxing
(School of Pharmacy, Xuzhou Medical College, Xuzhou, Jiangsu 221004)

Abstract The interactions between the two kinds of snake venom (cobra venom and viper venom) and 6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile at pH 7.6 were investigated by fluorescence quenching and 6-amino-4-(2-hydroxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5- carbonitrile (I) was studied in detail. The quenching constants KSV, binding constants K and sites n of I with venom were determined. The results indicated that both of interactions were static quenching procedures because of the formation of new compounds and there had a strong binding between them.