Spectral and Antimicrobial Activity of Picric Acid Charge-Transfer Complex With

Research Article

Spectral and Antimicrobial Activity of Picric Acid Charge-Transfer Complex With Modified Poly(Propylene Amine) Dendrimer

Corresponding author: Dr. Ivo Grabchev, Sofia University “St. Kliment Ohridski”, Faculty of Medicine, 1407 Sofia, Bulgaria, Tel: +35928161319; Email: i.grabchev@chem.uni-sofia.bg

Abstract

The charge transfer complex of poly(propylene amine) dendrimer peripherally modified with 1,8-naphthalimide units from sec- ond generation (as donor) and picric acid (as acceptor) has been synthesized and characterized by elemental analysis, IR and 1H-NMR-spectroscopy. Its basic photophysical characteristics have been investigated in organic solvents with different polarity. The electronic absorption spectra indicated that the charge transfer complex was formed with proton migration from the accep- tor to the donor followed by hydrogen bonding via N+–H• • •O−. The stoichiometry of the dendrimer charge transfer complex has been investigated by fluorescence spectroscopy and it was found to be 4:1 [PPA8(PA)4]. The complex was found to inhibit the growth of various Gram-positive and Gram-negative bacteria and yeasts.

Keywords: 1,8-Naphthalimide; Picric Acid; Dendrimer; Antibacterial Activity; Infrared Spectra; Fluorescence

Introduction

Dendrimers are a relatively new, hyperbranched and mono- disperse class of polymers, with well-defined molecular struc- ture Dendrimers combine the photophysical properties of low- and high molecular weight substances [1]. A great number of the same or different functional groups are located both in the branches and in the periphery, which gives many opportuni- ties for target modification of their properties [2-6]. Function- alising the dendrimers with photoactive groups expands the spheres of their applications [7,8]. Periphery modified den- drimers comprise many closely located chromophores which could be independent from each other or can interact. In the latter case the dendrimers acquire new properties defined as the “dendrimer effect”[9]. Poly(propylene amine) (PPA) and

polyamidoamine (PAMAM) are two commercial classes of den- drimers with particular application in various areas [1,9,10]. They are water-soluble, non-immunogenic and biocompatible.

Dendrimers might enhance the solubility of lipophilic drugs due to hydrophobic interactions, hydrogen bonding or elec- trostatic interaction between surface functional groups of the dendrimer and drug. Moreover, some of dendrimers show an- tibacterial and antifungal activity [11] and provide the oppor- tunity for complex therapy in which the dendrimers are not only the drug carrier but also an adjunctive component of the dosage form [12].

In recent years, charge-transfer (CT) complexes of different organic compounds have been studied intensively due to their

special type of interactions, accompanied by transferring of electrons from the donors to the acceptors. Charge transfer complexes play a central role in bactericides, fungicides, insec- ticides and various light-driven physical and chemical process- es [13-15]. Picric acid (PA) is a good electron acceptor and is known to form stable colored charge transfer complexes with many donors [16-18]. During the electron transfer this acidic acceptor forms an ion pair adduct [19]. CT complexes are ap- pealing also as potential antimicrobial agents. For example, CT complexes of a drug molecule may absorb in the visible range and thus lead to easy detection and estimation of the drug [20]. Nowadays, there is a need for new antimicrobials due to rise of resistance in many common bacterial pathogens [21]. By studying the antimicrobial activity of CT complexes new types of antimicrobial agents could be developed.

In this paper we describe the charge-transfer interaction be- tween peripherally modified poly(propylene amine) dendrim- er from second generation (as donor) and picric acid (as accep- tor). The stoichiometry of the reaction, IR and photophysical characterization of the obtained CT complex was discussed. In addition, the antimicrobial activity of the complex was inves- tigated.

Experimental part

Materials and methods

The synthesis and basic photophysical characteristic of PPA8 dendrimer from second generation modified with 1,8-naph- thalimide units were described earlier [22]. Infrared analysis was carried out using Infrared Fourier transform spectrometer (IRAffinity-1 “Shimadzu”) with the diffuse-reflectance attach- ment (MIRacle Attenuated Total Reflectance Attachment) at a 2 cm-1 resolution.All IR spectra were recorded in solid state. UV/vis spectrophotometric investigations were performed us- ing “Thermo Spectronic Unicam UV 500” spectrophotometer. The fluorescence spectra were taken on a “Cary Eclipse” spec- trophotometer. All spectra were recorded at concentrations of 1×10–6 mol dm–3 using 1 cm path length synthetic quartz glass cells. Organic solvents used in this study (methanol, eth- anol, 2-propanol, tetrahydrofuran and a dichlorethane) were of spectroscopic grade, and they were used as obtained from Sigma-Aldrich. Fluorescence quantum yield was determined on the basis of the absorption and fluorescence spectra us- ing Coumarin 6 as a reference (Φst = 0.78 in ethanol) [23]. 1H (600.13 MHz) spectrum was performed on an AVANCE AV600 II+NMR spectrometer. The measurements were carried out in CDCl3 solution at ambient temperature. The chemical shifts were referenced to a tetramethylsilane (TMS) standard.

Synthesis of the solid charge transfer complex of picric acid with modified poly(propileneamine) dendrimers from second gener- ation [PPA8(PA)4 ]

The solid charge transfer complex of picric acid with PPA8 from second generation was synthesized by reaction of PPA8 (0.29g, 1 mmol) dissolved in 10 ml chloroform with picric acid (0.13g, 6 mmol). The mixture was stirred at 400C for 60 min. The precipitate was filtrated, washed three times with chloro- form (3×10 ml), and then dried under vacuum at 400C.

Yield: 86%

1H-NMR (CDCl3, 600MHz ppm): 9.12 (s, 8H, Ar-PA, 7.62 (dd, J=5.86 Hz, J=5.81 Hz, 16H, ArH), 7.45 (dd, J=5.88 Hz, J=5.8 4 Hz, 16H, ArH), 7.24 (m, 16H, ArH), 4.14 (q, 16H, (OC)2NCH2),

1-70-1.54 (m, 4H, NH+) 1.44-1.01(m, 32H, CH2N< + 4H, >NCH-

2CH2CH2CH2N<), 0.97-0.64 (m, 24H, >NCH2CH2CH2N< + 4H,

>NCH2CH2CH2CH2N<).

Analysis: C160H140N26O44 (3128.3): Calcd. C 61.37, H 4.47, N

11.63; Found C 61.49, H 4.59, N 11.79.

Antimicrobial assay

The antimicrobial potential of the newly synthesized complex [PPA8(PA)4 ] was evaluated in vitro for its antibacterial and an- tifungal activities using disc agar-diffusion method [24]. The antibacterial activity was tested against Gram-positive bacte- ria Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Sarcina lutea ATCC 9341 and Micrococcus luteus ATCC 9631, and Gram-negative bacteria Pseudomonas aeruginosa NBIMCC 1390 (National Bank of Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria), Escherichia coli JM105 5α, Acineto- bacter johnsonii ATCC 17909 and Xanthomonas oryzae ATCC 35933. The antifungal activity was tested against the yeasts Saccharomyces cerevisiae ATCC 9763 and Candida lipolytica 76-18 (Institute of Microbiology collection, Sofia, Bulgaria). The CT complex was dissolved in DMSO in concentration 5 mg/ml and 20 µl of this solution was spotted on filter paper discs (6 mm in diameter) and applied onto nutrient agar plates with each freshly grown indicator culture. Standard discs of Gentamicin (antibacterial agent) and Nystatin (antifungal agent) were used as reference controls. Each treatment was performed in three replicates. After incubation of the plates for 24-48 h at 28 ± 2°C, the inhibition zone diameters (including disc), were measured.

Results

Charge transfer complex [PPA8(PA)4] formed from the inter- action between PPA8 and picric acid was synthesized by the mixture of both compounds in chloroform solution at 400C for 60 minutes. After cooling to room temperature, the yellow pre- cipitate was filtered and washed with chloroform (Scheme 1).

Scheme 1. Synthesis and chemical structure of [PPA8(PA)4] charge

transfer complex.

Photophysical characteristics of charge transfer complex [PPA8(PA)4]

Initial PPA8 dendrimer absorbs at UV region with maximum at λA = 326 nm and emits low (ΦF = 0.007) blue fluorescence emission with maximum at λF = 375nm. [22].

In Table 1 are summarized the basic photophysical character- istics of [PPA8(PA)4] measured in organic solvents of different

polarity. Charge transfer complex [PPA8(PA)4] absorbs in the λA = 337-354 nm region (ππ* transition) and an additional maximum at λA = 400-422 nm (n-π* transition) and have an in- tensive yellow color due to the complex formation (Table 1 and Figure 1). The absorption spectra provides evidence for the existence of new bands of the charge transfer complex which indicate an interaction associated with proton migration from the picric acid acceptor to the PPA8 donor followed by inter- molecular hydrogen bonding as presented in Scheme 1.

2-propanol Methanol Ethanol Tetrahydrofuran Dichlorethane
A (nm) 351

400

352

400

354

400

348

406

337

422

 (l mol-1 cm-1) 83000 82900 82300 85800 85900
F(nm) 518 525 524 515 511
νAF (cm-1) 9185 9361 9164 9318 10104
ΦF 0.09 0.02 0.03 0.11 0.14

Table 1. Photophysical properties of CT complex [PPA8(PA)4].

In all tested organic solvents the dendrimer complex emits yellow-green fluorescence with maxima at λF= 511-525 nm region, which is not typical for the free PPA8 dendrimer. This means that PPA8 dendrimer changes its fluorescence from blue to yellow-green after CT complex formation with PA. In this case, the yellow color of [PPA8(PA)4] is due to the dif- ferent polarization of 1,8-naphthalimide chromophoric sys- tem, compared to free PPA8 dendrimer. As seen from the data, [PPA8(PA)4] has negative solvatochromism (Table 1). The Stokes shift (νA -νF ) indicates the difference in the properties and structure of the [PPA8(PA)4] in the ground state S0 and the first exited state S1. The Stokes shift values are very large and they are in the 9164-10104 cm-1 region which is approximately two fold higher than the PPA8 values [25]. This indicates a de- stabilising effect of the PA on the planarity of dendrimer mol- ecule. The quantum yield of fluorescence (ΦF) is relatively low in all organic solvents and is in the region of ΦF= 0.02-0.14 but there is a tendency of enhancement.

Because the fluorescence spectroscopy is more sensitive com- pared to the absorption spectroscopy, we have used this tech- nique to investigate the interaction of picric acid with PPA8 dendrimer. As seen in Scheme 1, there is only tertiary amino groups in the core of peripherally modified with 1,8-naph- thalimide units PPA8 dendrimer, and these amino groups can act as a powerful electron donor. They can react with hydroxyl groups from picric acids and form hydrogen bonds

+N—H •••••O-. The titration profile of dendrimer PPA8 with picric acid is plotted in Figure 2. It is seen than 4 mol of picric acid react with 1 mol of dendrimer to form charge trans- fer complex [PPA8(PA)4] with stoichiometry 4:1.

Cite this article: Ivo Grabchev. Spectral and Antimicrobial Activity Of Picric Acid Charge-Transfer Complex With Modified Poly(Propylene Amine) Dendrimer.

J J Organic Chem . 2016, 1(1): 002.

Figure 1. Absorption spectra of [PPA8(PA)4] in : ethanol (1), metha- nol (2), 2-propanol (3), tetrahydrofurane (4), chloroform (5).

Infrared spectra of [PPA8(PA)4] complex

Stretching and deformation vibrations of the main functions in the infrared region of the spectra of PPA8 dendrimer and [PPA8(PA)4] are summarised in Table 2. As can be seen, the characteristic frequencies for the mains groups of PPA8 are approximately identical with these for [PPA8(PA)4]. Similar results have been described recently [25].

Figure 2. Fluorescence titration curve for [PPA8(PA)4] in chloroform

solution (PPA8 c= 1×10-6 mol dm-3).

That means than the influence of picric acid is not so strong to cause a change in the frequency of oscillation of the charac- teristic groups. This is especially well seen in the region 1698 and -1655 cm-1, where absorb both carbonyl groups ν(C=O) [26,27].

Figure 3 shows the difference in the spectra of PPA8 and its complex [PPA8(PA)4]. The most significant difference is ob- served in the region 1200-1400 cm-1, where are the charac- teristic bands of nitro (-NO2) and C-N groups [28]. The asymmetric stretching vibration of the –NO2 group is sensitive to the polar influences and the electronic states. In the spectrum of [PPA8(PA)4] a specific doublet appears at 1564-1555 cm, and new intensive bands at 1313 cm-1 are characteristic to the nitro groups of picric acid. The C-N bond showed stretch- ing vibration absorption bands at 1363, 1337,1262 and 844 cm-1. C–NO2 stretching vibration are observed at 1078 and 910 cm−1. A broad doublet at the spectrum of [PPA8(PA)4] has been detected with maximum at ca. 2646 cm-1 which was not observed at PPA8 spectrum. Probably It can be ascribed to the stretching vibrations of the picric acid hydrogen bonds (+N—H •••••O-). ( Figure 4).

NMR spectral characterization of [PPA8(PA)4] complex

The chemical structure of [PPA8(PA)4] was confirmed by 1H-NMR spectroscopy in chloroform. The singlet peak observed at δ 9.12 has been assigned to aromatic protons of the picrate moiety in the complex. The aromatic protons from 1,8-naphhthalimide units are at δ(ppm) 7.62, δ 7.45 and δ

7.24. Aliphatic protons appear as multiplets in the areas of:δ (ppm) 4.14,δ 144-1.01 and δ 0.97-0.64. The multiplets at δ(ppm) 1.70-1.54 are due to the protonated nitrogen atoms (NH+). The characteristic signals for aromatic and aliphatic protons of PPA8 dendrimer structure from the complex are shifted to the lower δscale as compared to the initial PPA8 den- drimer [22]. Also, the intensities of the aromatic signals were significantly affected by the complexation process and the ac- companying changes in the structural configuration. The char- acteristic proton peaks from hydroxyl group of picric acid atδ

= 11.94 ppm, [29], was absent in the spectrum of the [PPA8(- PA)4] complex.

PPA8 (cm-1) [PPA8(PA)4] (cm-1)
C-H arom 3068 3073
CH2 aliph 2958

2874

2972

2866

AS

C=O

1696 1698
S

C=O

1654 1655
C=C arom 1624

1595

1609

1589

CH2 aliph 1437 1436
CNC 1350

1173

1362

1164

C-H arom 844

774

844

774

Table 2. Infrared spectra of 4PPA8 and its complex [PPA8(PA)4].

Cite this article: Ivo Grabchev. Spectral and Antimicrobial Activity Of Picric Acid Charge-Transfer Complex With Modified Poly(Propylene Amine) Dendrimer.

J J Organic Chem . 2016, 1(1): 002.

Figure 3. FTIR spectra of PPA8 and [PPA8(PA)4]

Microbiological investigations

The antibacterial activity of the newly synthesized complex was tested against eight bacterial strains, and the antifungal activity against two yeast strains. The [PPA8(PA)4] complex (100 µg/disc) demonstrated inhibitory activity against the growth of six of the test bacteria with zones of inhibition in the range 10-12 mm (Figure 5). In comparison with standard drug, the complex exhibited lower or comparable activity. Gram-negative strains E. coli and P. aeruginosa were resistant towards the studied complex. The complex exerted good anti- fungal activity against the test yeasts, which were found resis- tant towards the control antifungal agent nystatin. Therefore, the new dendrimer complex has potential in developing new antimicrobial agents.

Figure 4. FTIR spectra of PPA8 and [PPA8(PA)4].

Figure 5. Inhibition of the growth of some model bacteria and yeasts by [PPA8(PA)4] complex. G/Ns, gentamicin/nystatin used as a stan- dard antibacterial/antifungal agent.

Conclusion

Fluorescence charge transfer complex [PPA8(PA)4] from mod- ified PPA dendrimers with 1,8-naphthalimide units and picric acid has been synthesized and characterized by electronic (ab- sorption and fluorescence), FT-IR and 1H-NMR spectroscopy. The data from elemental analysis, NMR and FT-IR spectrosco- py indicated that the amine and phenolic groups are involved in the formation of the charge transfer complex between PPA8 and PA. The main photophysical characteristics have been in- vestigated in organic solvents of different polarity and it was shown that [PPA8(PA)4] dendrimer emits yellow-green fluo- rescence. The formation of charge transfer complex of PPA8 and PA was investigated spectrophotometrically in chloro- form at room temperature by fluorescence spectroscopy and it was found a complex formation with 1:4 stoichiometry. The [PPA8(PA)4] complex exhibited antibacterial and antifungal activity against various species of bacteria and yeasts. Investi- gation of the antimicrobial activity of such type of dendrimer complexes would expand the potential biological application of the dendrimers.

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