Abstract: A praseodymium salt of 12-tungstophosphate acid, Pr[PW12O40], was synthesized using ion exchange and double decomposition method. Then its 2e and 4e electrochemically reduced heteropoly blues, PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40], were synthesized using control-potential electrolysis reduction method. These compounds were characterized with FT-IR, UV-vis, polarography, ESR and XPS. The α-Keggin type structure was kept in the heteropoly blues PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] but the electronic density of heteropoly anion increased after electrochemically reduction. Heteropoly blues PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] showed relatively high in vitro anti-HIV-1 activity and relatively low cytotoxicity on MT-4 cells compared to some other anti-HIV-1 compounds. Because heteropoly blues possess higher negative electronic charges than their non-reduced parents, they strongly interact with HIV-1, MT-4 cells and macrophages and effectively suppress fusion, infection and proliferation of HIV-1.
Introduction
Polyoxometalate (including heteropoly oxometalate and isopoly oxometalate) is a large class of inorganic transition metal oxygen anion clusters (Pope, 1983). Because a heteropoly anion consists of one central atom (P, Si, Ge, etc.) surrounded by some coordinate atoms (W, Mo, V, etc.), hereropoly anions usually possess cage-like molecular structures with some unique properties. In general, the coordinate atoms in heteropoly oxometalates exhibit in the highest valence (the d0 species: WVI, MoVI, VV, etc.). The coordinate atoms in some heteropoly anions can be reduced to form the electrochemically reduced species. Because most of the electrochemically reduced species look blue no matter what colors their non-reduced parents are, these electrochemically reduced species are also called as heteropoly blues. Heteropoly blues usually retain the general structures of their non-reduced parents, but have some special properties that their oxidized parents do not have (Wang et al., 1992, 1996; Yuan et al., 2003). Thus heteropoly blue is a large potentially important group of mixed-valence heteropoly oxometalates.
Heteropoly oxometalates are remarkable for their molecular and electronic structural diversity and they are promising in catalysis and material filed (Liu et al., 2000, 1996). In recent years, the application of heteropoly oxometalates in medicine filed has made great progress (Rhule et al., 1998; Liu et al., 2005). As early as the acquired immune deficiency syndrome (AIDS) caused by human immunodeficiency virus type 1 (HIV-1) was discovered, heteropoly oxometalates have been studied as anti-AIDS inorganic compounds (Dagani, 1986). (NH4)17Na[NaSb9W21O86]•14H2O (HPA-23) was the first heteropoly oxometalates applied to clinical trials for the AIDS patients (Dormont et al., 1985; Balzarini et al., 1986). But owing to its higher toxicity, HPA-23 could not be put on the market as a medicine (Moskovitz et al., 1988). However, after Keggin structure heteropoly oxometalates (especially K7[PW10Ti2O40]•6H2O (PM-19)) were found to possess low toxicity and high anti-HIV activity from 1990s (Inouye et al., 1990; Hill et al., 1990), the study on the anti-HIV effect over heteropoly oxometalates becomes a popular research field again. The counter cations also influence the anti-virus activity of heteropoly oxometalates. We have reported that rare earth salts (especially Pr salt) of [PW10Ti2O40]7¯ possess higher in vitro anti-HIV-1 activity than K7[PW10Ti2O40]•6H2O (PM-19) (Liu et al., 1996). Moreover, (iso-PrNH3)6H[PW10Ti2O38(O2)2]•9H2O (PM-523) has also been reported as a broad spectrum antimyxovirus agent (Shigeta et al., 1996). These results indicate that Pr ion is a good counter cation for heteropoly oxometalates to improve their anti-virus activity.
Although a lot of heteropoly oxometalates had been investigated for their anti-HIV activity (Rhule et al., 1998; Inouye et al., 1990; Hill et al., 1990; Liu et al., 1996; Shigeta et al., 1996; Barnard et al., 1997; Shigeta et al., 2003; Weeks et al., 1992; Kim et al., 1994), the anti-HIV activity of heteropoly blues was not carefully investigated till now except for few reports from our group in Chinese (Liu et al., 2004).
Based on the experiences of anti-HIV research from one author (Y. Zeng) (Zeng et al., 1984; Chen et al., 2002) and the knowledge of heteropoly blues from one author (E. Wang) (Wang et al., 1992, 1996; Yuan et al., 2003), we synthesized heteropoly blues PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] in the first time and investigated their in vitro anti-HIV-1 activity in the present study.
Materials and Methods
Materials
The cooperation research for investigating in vitro anti-HIV-1 activity
over electrochemically reduced heteropoly blues was started in 2002. The synthesis
of heteropoly compounds was conducted in Institute of Polyoxometalate Chemistry,
Department of Chemistry, Northeast Normal University (Changchun, China). The
anti-HIV-1 experiments were performed in Department of Tumor Virus and HIV,
Institute of Virology, Chinese Academy of Preventive Medicine (Beijing, China).
HIV-1 was kindly provided by Prof. L. Montagnier, Institut Pasteur (Paris, France).
3’-azido-3’-deoxythymine (AZT) and Dextran surface (DS) were obtained
from Tsukuba Laboratories, GlaxoSmithKline Co. (Tsukuba, Japan).
α-H3[PW12O40] was prepared according to the method in literature (Rocchiccioli-Deltcheff et al., 1983), its structure was confirmed by 31P NMR and polarogram.
Pr[PW12O40], a praseodymium salt of α-H3[PW12O40], was prepared using an ion exchange and double decomposition method (Wang et al., 1996; Tsigdinos, 1974). A portion of 1.0 g of α-H3[PW12O40] was solved in 50 mL water and then a calculated amount of Pr2(CO3)3 powder was added to the solution with strong stirring. After the CO2 gas had been eradiated, the filtrate was obtained by filtering off a small amount of solid by-product. Then, the filtrate was put in a dry box for five days to obtain the product (0.9 g). Element analyses and TG/DTA proved that the chemical composition of the product was Pr[PW12O40]•18H2O.
Heteropoly blues, PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40], were prepared using a control-potential electrolysis reduction method (Wang et al., 1996). A solution of 1 g of Pr[PW12O40]•18H2O in 100 mL 0.1 M HCl solution, was placed in an electrochemical cell and reduced to a degree corresponding to the transfer of two or four electrons into per Keggin unit (Econtrol = -0.40 V (2e), -0.65 V (4e) (vs. SCE)). The reduction extent was indicated by a coulometer. The cathode liquid was stirred with nitrogen stream to purge oxygen. After the electrochemical reduction, the solution was set in a vacuum desiccator. The blue crystal appeared one week later and then the product was recrystallized twice with 0.1 M HCl. The composition and the number of crystal water in the final products were analyzed with elemental analyses and TG-DTA. The chemical compositions of two electrons and four electrons reduced heteropoly blues were PrH2[PW12O40]•20H2O and PrH2[PW12O40]•20H2O, respectively.
K7[PW10Ti2O40]•6H2O (PM-19) and (NH4)17Na[NaSb9W21O86]•14H2O (HPA-23) were synthesized using the methods in the literatures (Domaille et al., 1983; Fischer et al., 1976; Michelon et al., 1980). Both of them were confirmed by FT-IR spectra and element analyses.
Instrumentation
Inductively Coupled Plasma (ICP) analyses were carried out using a Thermo
Jarrell Ash IRIS/AP instrument. TG/DTA were carried out using a Shimadzu TGA-50
instrument. FT-IR spectra were recorded on a JASCO FT/IR 7000 spectrometer using
KBr pellets under atmospheric conditions. UV-vis spectra were recorded on a
Beckman-DU70 spectrometer in aqueous solutions. Polarograms were recorded on
a Sargernt Model XV instrument using a dropping mercury electrode and standard
H cell with a saturated calomel electrode. ESR spectra were recorded on a JES-FE-3AS
spectrometer. XPS spectra were collected at a chamber pressure of 10-9
Torr using a Phi-5500 ESCA spectrometer employing Mg Kα radiation
(1253.6 eV).
Measurement of Anti-HIV-1 Activity
The MT-4 cell lines were maintained in RPMI-1640 medium supplemented with
10% fetal calf serum, 100 μg mL-1 penicillin and 100 μg
mL-1 streptomycin.
The evaluation of anti-HIV activity for every compound was based on a MTT method as reported in the literature (Pauwels et al., 1988). The living cells can reduce the yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a blue formazan but the dead cells do not possess this capability. Thus the number of living cells could be calculated by the change of optical density. MT-4 cells were infected with HIV-1 at a multiplicity of infection (moi) of 0.01 at 37°C for 1 h. Then the inflected MT-4 cells were dispensed into wells of 96-well microtiter trays and incubated with individual test compounds in a total volume of 200 μL at 37°C for 5 days. The optical density of the solution was then read on a Dynatech MR700 Microelisa reader, using a test wavelength of 540 nm and a reference wavelength of 690 nm. The percentage protection was calculated using the following formula:
Where (ODT)V, (ODC)V and (ODC)M indicated the absorbance of the test sample (treated by virus and compound), the virus infected sample (no compound) and the mock infected control sample (no virus and no compound), respectively. The median effective concentration (E50) was expressed as the concentration that achieved 50% protection of virus infected cells from virus-induced destruction.
The cytotoxicity of every compound on non-infected MT-4 cells was examined by the MTT method in parallel with the anti-HIV-1 experiment. The number of living cells was examined after 5 days incubation in the presence of different concentrations of every compound without HIV infection. The median cytotoxic concentration (CC50) was determined from the dose-response curve.
In order to check the reliability of the data obtained form MTT method, the cytotoxicity of every heteropoly compound was also evaluated by a Cytopathic Effect (CPE) inhibition method (Sidwell et al., 1971). MT-4 cells were dispensed into wells of 96-well microtiter trays at various concentrations of heteropoly oxometalates. After incubated with growth medium at 37°C for 5 days, the complete viral CPE was observed by light microscopy. Morphological changes due to the compounds cytotoxicity were graded on a scale of 1-4: grade 1 (-) was defined as no changed MT-4 cells; grade 2 (+) was defined that the percentage of changed cells was less than 25%; grade 3 (++) was defined that the percentage of changed cells was more than 25% but less than 75%; grade 4 (+++) was defined that the percentage of changed cells was more than 75%.
The Selective Index (SI) that indicates the anti-HIV-1 activity over every compound was calculated corresponding to the ratio CC50/EC50.
Results and Discussion
Characterization of Heteropoly Blues
Keggin type is one of the most popular structures for heteropoly anions.
As shown in Fig. 1a Keggin structure heteropoly anion consists
of a central XO4 tetrahedron (such as PO4) surrounded
by twelve MO6 octahedra (such as WO6). The twelve MO6
octahedra comprise four groups of three edge-shared octhedra, the M3O13
triplet, which have a common oxygen vertex connected to the central XO4
tetrahedron.
| Fig. 1: | The structure of Keggin type heteropoly anion |
| Table 1: | Peaks and their assignments in FT-IR spectra of heteropoloy compoundsa) |
| a) Measured at room temperature using Kbr pellet method | |
| Table 2: | Bands and their assignments in UV-vis spectra of heteropoly compoundsa) |
| a) Measured at room temperature in aqueous solutions, b) Molar extinction eoefficients listed in parenthesis | |
The oxygen atoms in this structure fall into four classes of symmetry-equivalent oxygens: X-Oa-(M)3; M-Ob-M, connecting two M3O13 units by corner sharing; M-Oc-M, connecting two M3O13 units by edge sharing; and M = Od (Pope, 1983). Among the heteropoly compounds used in this study, the heteropoly anions in α-H3[PW12O40], Pr[PW12O40] and K7[PW10Ti2O40]•6H2O (PM-19) are Keggin type heteropoly anions but the heteropoly anion in (NH4)17Na[NaSb9W21O86]•14H2O (HPA-23) is not a Keggin type heteropoly anion.
The peaks and their assignments in IR spectra of Pr[PW12O40], PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] are shown in Table 1. PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] showed the four characteristic vibrations for the Keggin type heteropoly anions (vas(P-Oa), vas(W = Od), vas(W-Ob-W) and vas(W-Oc-W)) with only slightly changes compared to those of Pr[PW12O40]. This indicates that the reduced heteropoly blues PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] still retain the Keggin structure with a little distortion. It is noteworthy that the frequencies of all characteristic vibrations shifted to high frequency regions from Pr[PW12O40] to PrH2[PWVI10WV2O40] and from PrH2[PWVI10WV2O40] to PrH4[PWVI8WV4O40]. The electronic density in the heteropoly anion increased due to the induction of electrons during the reduction process, which caused the increase of the strengths of P-Oa, W = Od, W-Ob-W and W-Oc-W bonds. Thus the characteristic vibration peaks of Keggin type Pr[PW12O40] shifted to the high frequency regions in IR spectra after electrochemical reduction.
The data of UV-vis spectra of Pr[PW12O40], PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] are shown in Table 2. Keggin structure heteropolytungstate anions have characteristic absorption bands at about 200 and 260 nm in UV region corresponding to charge transfers of Od-WVI and Ob,c-WVI (Pope, 1983). As shown in Table 2, both PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] showed these bands whose positions were quite similar to that of Pr[PW12O40]. These results are coincided with the results from IR spectra that the Keggin type structure was kept in the heteropoly blues PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] after electrochemical reduction.
The appearance of a strong absorption bond at about 740 nm in visible region is a signal for the formation of heteropoly blue. This is the reason why the electrochemical reduced heteropoly compounds look blue. This band can be assigned to Intervalence Charge-transfer (IVCT) from WV-Ob-WVI to WVI-Ob-WV according to its strength and position (Pope, 1983). Pr[PW12O40] only showed a weak broad absorption band in visible region due to the absorption of Pr3+ cation (not shown in Table 2) and did not show any absorption bands at about 740 nm. On the other hand, both PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] showed strong a bsorption bands at about 740 nm, which indicates that heteropoly blues were formed after electrochemically reduction of Pr[PW12O40].
| Table 3: | Polarograms data of various heteropoly compoundsa) |
| a) Measured in 0.1 N HCI solution at room temperature | |
The band intensity of PrH4[PWVI8WV4O40] at about 740 nm is stronger than that of PrH2[PWVI10WV2O40] due to the induction of two extra electrons in PrH4[PWVI8WV4O40].
The half wave potentials in polarograms of α-H3[PW12O40], Pr[PW12O40], PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] are shown in Table 3. Both the wave shapes and the half wave potentials in polarograms are quire similar among α-H3[PW12O40], Pr[PW12O40], PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40]. All of them showed three waves corresponding to one-electron, one-electron and two-electrons reduction processes. In general, the wave shapes and the half wave potentials in the polarograms are quite different among α-isomer, β-isomer and γ-isomer of Keggin type heteropoly anions (Biquard et al., 1971). No significant changes were observed in polarograms after the process of ion exchange and double decomposition and after the process of electrochemical reduction. Therefore, The α-isomer of Keggin structure is concluded to be kept in Pr[PW12O40], PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] as the starting heteropoly compound α-H3[PW12O40].
Pr[PW12O40] did not show any ESR signals both at room temperature and at 77 K. As for PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40], no ESR single could be detected at room temperature because of the strong spin-lattice relaxation resulting from both the electron delocalization and the thermal excited electron jumping. However, the ESR signals with isotropic line shapes could be observed at 77 K for both PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] at g = 1.83. Because this g value is the characteristic position of WV ion, the electrons introduced into the heteropoly blues mainly localized in the orbit of WV ions with little delocalization at low temperature like MoV ions containing heteropoly blues (Sanchez et al., 1982).
The binding energy of electrons in inner shell of P2p and O1s changed little in the XPS spectra among Pr[PW12O40], PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40]. This indicates the structures of three samples closely resemble with each other. The signals of W4f7/2 in PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40] were broad and deformed compared to that in Pr[PW12O40]. This phenomenon could be explained by the additive effect of the splitting peaks for both WV and WVI in the heteropoly blues. After decomposed the W4f7/2 singles in PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40], the ratios of the peak area of WV to WVI were almost 2:10 for PrH2[PWVI10WV2O40] and 4:8 for PrH4[PWVI8WV4O40], respectively. These results strongly suggest that the reduced heteropoly blues PrH2[PWVI10WV2O40] has two WV ions and PrH4[PWVI8WV4O40] has four WV ions in their structures as was expected.
As described in above, the results of FT-IR, UV-vis, polarography, ESR and XPS proved that the electrochemically reduced praseodymium salts of 12-tungstophosphate acid, PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40], have been synthesized successfully in this study.
| Table 4: | Cytotoxicith and anti-HIV-1 activity of various compoundsa) |
| a) Data represent mean values for three experiments. Cytotoxicity and anti- HIV-1 acitivity were measured by the MTT method. b) CC50: the median cytotoxic concentration of compound for achieving 50% protection of HIV infected MT-4 cell from virus-induced destruction. d) SI: the ratio of CC50 to EC50 | |
| Table 5: | The cytotoxicity on MT-4 cell of various heteropoly oxometalatesa) |
| a) Data represent mean values for three experiments. Morphological change of MT-4 cell was observed by light microscopy after 5 days incubation in medium with heteropoly oxometales. b) -: no cell changed cells in the range from 25 to 75%; +++: percentage of changed cells >75%. c) Maxnon: the maximum non-cytotoxic dose | |
Cytotoxicity on Mt-4 Cells and In vitro Anti-hiv-1 Activity of Heteropoly
Oxometalates
The cytotoxicity on MT-4 cells and in vitro anti-HIV-1 activity evaluated
by the MTT method over various compounds are shown in Table 4.
Keggin type heteropoly oxometalates showed their median cytotoxic concentration
(C50) at a range from 260 to 320 μg mL-1. On the
other hand, the non-Keggin type heteropoly oxometalates HPA-23 showed a low
C50 value of 42 μg mL-1, which indicates that HPA-23
possesses a high cytotoxicity on MT-4 cells. These results are consistent with
the results reported in the literatures. α-H3[PW12O40]
and Pr[PW12O40] showed their SI values at 35 and 44, respectively.
This suggests that Pr3+ ion possesses the ability to improve the
anti-HIV-1 activity of [PW12O40]3¯ heteropoly
anion. Because the SI values of Pr[PW12O40], PrH2[PWVI10WV2O40]
and PrH4[PWVI8WV4O40]
are 44, 66 and 70, the anti-HIV-1 activity is in the order of Pr[PW12O40]
<PrH2[PWVI10WV2O40]<PrH4[PWVI8WV4O40].
A higher negative charges that a heteropoly anion possesses, a higher anti-HIV
activity could be expected. PrH2[PWVI10WV2O40]
and PrH4[PWVI8WV4O40]
showed higher SI values than that of PM-19, which indicates that heteropoly
blues PrH2[PWVI10WV2O40]
and PrH4[PWVI8WV4O40]
are effective heteropoly oxometalates for anti-HIV-1. DS showed low cytotoxicity
on MT-4 cells and its SI value was similar to those of PrH2[PWVI10WV2O40]
and PrH4[PWVI8WV4O40].
On the other hand, AZT showed a high SI value of 1000 but its cytotoxicity was
very high. Therefore, it seem that the mechanism of anti-HIV over heteropoly
oxometalates is similar to the mechanism over DS but is different from the mechanism
over AZT.
The cytotoxicity on MT-4 cells over various heteropoly oxometalates observed by the CPE effect using light microscopy are shown in Table 5. All Keggin type heteropoly compounds (PM-19, Pr[PW12O40] and its reduced species) showed low cytotoxity on MT-4 cells and their maximum non-cytotoxic doses were higher than 150 μg mL-1. On the other hand, the non-Keggin structure heteropoly oxometalates HPA-23 showed high cytotoxicity on MT-cells, its maximum non-cytotoxic dose was lower than 50 μg mL-1. Comparing the results in Table 4 and Table 5, the cytotoxicity of various heteropoly compounds did not show significant difference between using the MTT method and using the CPE inhibition method. This indicates that the MTT method is reliable for evaluating the cytotoxicity on MT-cells and in vitro anti-HIV-1 activity over various compounds.
As shown in Table 4 and 5, two heteropoly blues prepared in this study, PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40], showed higher in vitro anti-HIV-1 activity and lower cytotoxicity on MT-4 cells comparing to the reported heterpoly compound anti-HIV-1 agents. Moreover, although the anti-HIV-1 activity in vitro of heteropoly blues were lower than that of AZT, heteropoly blues showed much lower cytotoxicity on MT-cells comparing to AZT.
Several groups have studied the mechanism of the anti-HIV effect over heteropoly oxometalates (Take et al., 1991; Inouye et al., 1995; Judd et al., 2001; Ni et al., 1995; Witvrouw et al., 2000). Although the mechanism of anti-HIV activity over heteropoly oxometalates has not been fully elucidated, two mechanisms including inhibition of binding/fusion process and inhibition of reverse transcriptase, are responsible for their antiviral activity. The anti-HIV activity over a heteropoly oxometalate is determined by many factors, such as shape, size, surface charge, species of heteropoly oxometalates and so on. From the views of all reported mechanisms, the high negative charge of heteropoly anions is one of the most important factors for improving the anti-HIV activity: high negative charge could promote the ability of heteropoly anions to block virion absorption to membrane receptors to inhibit the fusion process of HIV infection (Take et al., 1991; Inouye et al., 1995); high negative charge could improve the ability of heteropoly anions to bind to the cationic pocket on the “hinge” region of the flaps to cover the active site of HIV protease (Judd et al., 2001); high negative charge could increase the accumulated ability in macrophages of heteropoly anions to suppress HIV proliferation (Ni et al., 1995; Witvrouw et al., 2000).
Heteropoly blues possess the same structure and the same composed elements as their non-reduced parent heteropoly oxometalates. The difference between heteropoly blues and their non-reduced parents is just that heteropoly blues possess higher negative charges. Therefore, it seem that the higher negative charges caused higher in vitro anti-HIV-1 activity of heteropoly blues.
We think heteropoly blues could provide us two ideas for anti-HIV research. One idea is preparing the heteropoly blues of an effective heteropoly oxometalate because heteropoly blues possess higher anti-HIV activity than their non-reduced parents. Another idea is studying the anti-HIV mechanism of heteropoly oxometalates using heteropoly blues and their non-reduced parents because heteropoly blues have same characters (shape, size and composed elements) as their non-reduced parents except that heteropoly blues have higher negative charges.
In conclusion, we successfully synthesized the electrochemically reduced praseodymium salts of 12-tungstophosphate acid (PrH2[PWVI10WV2O40] and PrH4[PWVI8WV4O40]) for the first time. Because the prepared electrochemically reduced heteropoly blues remain the structures of their non-reduced parent heteropoly oxometalates but possess higher negative charge comparing to their non-reduced parent heteropoly oxometalates, they showed relatively high in vitro anti-HIV-1 activity and relatively low cytotoxicity on MT-4 cells.