International Journal of Biological Macromolecules 63 (2014) 83–91
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis and antifungal properties of (4-tolyloxy)-pyrimidyl-␣-aminophosphonates chitosan derivatives Yukun Qin a , Ronge Xing a , Song Liu a , Huahua Yu a , Kecheng Li a , Linfeng Hu a,b , Pengcheng Li a,∗ a b
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China School of Chemistry & Chemical Engineering, Henan Institute of Science and Technology, Xinxiang 453003, China
a r t i c l e
i n f o
Article history: Received 12 September 2013 Received in revised form 17 October 2013 Accepted 21 October 2013 Available online xxx Keywords: ␣-Aminophosphonate chitosan Synthesis Antifungal activity
a b s t r a c t A novel class of ␣-aminophosphonate chitosan derivatives was investigated. These chitosan derivatives consist of (4-tolyloxy)-pyrimidyl-dimethyl-␣-amino-phosphonate chitosan (␣-ATPMCS) and (4-tolyloxy)-pyrimidyl-diethyl-␣-aminophosphonate chitosan (␣-ATPECS). Their structures were well defined. Antifungal activity of them against some crop-threatening pathogenic fungi was tested in vitro. The derivatives were found to have a broad-spectrum antifungal activity that was obviously enhanced compared with chitosan. At 250 mg/L, both ␣-ATPMCS and ␣-ATPECS even inhibited growth of Phomopsis asparagi (Sacc.) (P. asparagi) and Fusarium oxysporum (F. oxysporum) at 100%, which was even stronger than polyoxin whose antifungal index was 37.2% and 32.1%, respectively. Additionally, the initial mechanism of the chitosan derivatives in F. oxysporum model was studied. It was found that the derivatives may have an effect on membrane permeability of the fungi. The results demonstrated the derivatives may serve as attractive candidates in crop protection. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitosan (poly--(1,4)-d-glucosamine) is an abundant biopolymer obtained from chitin. As an important renewable biopolymer, it attracts much attention in such fields as pharmacology, agriculture, materials, and food due to its good biocompatibility, biodegradability, and non-toxicity [1–5]. Antimicrobial activities of chitosan have been widely explored in vitro and in vivo. It was proved that chitosan can control a variety of bacteria and fungi [6,7]. However, chitosan is generally insoluble in water and most organic solvents for its strongly hydrogenbonding network structure [8]. Additionally, its antimicrobial activity is relatively lower than that of commercial antimicrobial agents. Thus, the use of chitosan as an antimicrobial agent has been restricted. Combinations of high molar mass polymers and low molar active molecules normally can take synergistic advantage of their unique properties, including the mechanical properties of polymers and bioactive properties of the small molecules. Thus, chemical modifications are an efficient approach in improving both water solubility and antimicrobial activity of chitosan [9–11]. Many
∗ Corresponding author at: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. Tel.: +86 532 82898707; fax: +86 532 82968951. E-mail addresses: [email protected] (S. Liu), [email protected], [email protected] (P. Li). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.023
attempts have been made to create new chitosan derivatives with an enhanced solubility and antimicrobial activity [12,13]. These derivatives include alkylation, acylation, quaternization, thiosemicarbazones chitosan and so on. ␣-Aminophosphonates, a kind of natural amino acid analogs, is an important class of compounds acted such as enzyme inhibitors [14], pharmacological agents [15], and pesticides [16]. In previous study, we reported the synthesis of 2-(␣-arylaminophosphonate)-chitosan via introduction ␣aminophosphonates group into chitosan backbone [17]. The obtained ␣-aminophosphonates chitosan showed a significant inhibiting effect on the investigated fungi compared with origin chitosan. However, their structures were not fully characterized (only in FT-IR) and the antifungal results were also not so satisfactory for them to be served as fungicidal candidates. Therefore, it was necessary to discover more effective derivatives of this kind via further chemical optimization. Heterocyclic compounds particularly six-membered compounds have occupied a prominent place among various classes of organic compounds for their diverse biological activities. Among a wide variety of heterocycles, pyrimidines are the integral part of nucleic acids and have been associated with a number of biological activities. They have played an important role in pesticide chemistry [18,19]. Fungicides with pyrimidines such as cyprodinil, ferimzone, mepanipyrim are widely used in crop protection. On the basis of the literature, we made the further assumption that combination of the pyrimidines
84
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Scheme 1. Synthesis of 4-(pyrimidyl-2-yloxy)benzaldehyde (a) and (4-tolyloxy)-pyrimidyl-␣-aminophosphonates chitosan (b).
moiety with ␣-aminophosphonates chitosan may enhance their antifungal activity. In this study, with the aim to develop more effective ␣-aminophosphonates chitosan derivatives, we focused our attention on grafting fungicidal pyrimidyl molecules that are known to inhibit crop-threatening pathogenic fungi on ␣aminophosphonates chitosan framework. To validate the hypothesis that a pyrimidyl moiety is required for improving the antifungal activity, the inhibitory effects of new obtained chitosan derivatives were tested in vitro. 2. Experimental 2.1. Materials Chitosan, with 87% degree of deacetylation and averagemolecular weight of 230 kDa, was supplied from Qingdao Baicheng Biochemical Corp. Polyoxin, 10% wettable powders, was purchased from Kaken Pharmaceutical Co., Ltd. 2-Chloropyrimidine was purchased from Chengdu Best Reagent Co., Ltd. (China). 4-Hydroxybenzaldehyde was purchased from Shanghai Shiyi Chemical Reagent Co., Ltd. (China). Other commercial chemical reagents used in the experiment were obtained from Sinopharm Group Chemical Reagent Co. Ltd. All of the regents were analytical grade and used without further purification. 2.2. Fungal strains The crop pathogenic fungi tested were obtained from Qingdao Academy of Agricultural Sciences. There were a total of four fungi strains: Rhizoctonia solani Kühn (R. solany), Phomopsis asparagi (Sacc.) (P. asparagi), and Fusarium oxysporum (F. oxysporum). Cultures of each of the fungi were maintained on potato dextrose agar (PDA) medium and were stored at 4 ◦ C. 2.3. Characterization Fourier transform infrared (FTIR) spectrum was measured in the 4000–400 cm−1 . Solid-state 13 C CP/MAS NMR spectra were performed on a Bruker AC-300 spectrometer. A ramped contact-power cross-polarization (CP) pulse sequence was applied and a doubleair-bearing Magic Angle Spinning (MAS) probe with 7 mm external diameter sample rotors was employed. Time domain size is 2048. The number of scans is 1000. 31 P NMR was recorded on samples
with a single pulse sequence model used a Bruker AC-300 spectrometer. The elemental analysis (C, H, and N) was carried out using a Vario EL-III elemental analyzer. The percentages of carbon, hydrogen and nitrogen were estimated and the degree of substitution (DS) of the chitosan derivatives was calculated on the basis of the percentage. X-ray diffraction (XRD) measurement of the powder samples were performed with a D8 Advance diffractometer (Bruker) with Cu target ( = 0.154 nm) at 40 kV and the scanning scope of 2 was 5–50◦ . Differential scanning calorimetry (DSC) was performed using the Pyris Diamond DSC (Perkin Elmer). The samples were heated from 50 ◦ C to 230 ◦ C under 10 ◦ C/min. Nitrogen gas was used in the experiment to confirm thermal behavior. The surface morphology of the samples was analyzed by scanning electron microscopy by using KYKY-2800B SEM. 2.4. Synthesis of 4-(pyrimidyl-2-yloxy)benzaldehyde A mixture of 4-hydroxybenzaldehyde (50 mmol, 6.1 g) and potassium carbonate (60 mmol, 8.28 g) was stirred in a 250 mL round bottomed flask with 100 mL DMF for half an hour at room temperature. Then 2-chloropyrimidine (50 mmol, 6.1 g) was added and the reaction mixture was stirred for another 10 h at 100 ◦ C until the 2-chloropyrimidine disappeared (TLC control). The residue was dissolved in ethyl acetate and washed with water. The combined organic layer was dried over MgSO4 and concentrated in vacuo to give the crude yellow oil. The residue was purified by column chromatography on silica gel and the product was obtained with 90.0% yield. 1 H NMR (600 MHz, DMSO-d6 , ı): 10.00 (s, 1H, CHO), 8.65 (d, 2H, pyrimidyl-H), 7.97 (d, 2H, Ph-H), 7.33 (d, 2H, Ph-H), 7.32 (t, 1H, pyrimidyl-H). 2.5. Synthesis of (4-tolyloxy)-pyrimidyl-˛-aminophosphonates chitosan Chitosan (20 mmol), the corresponding 4-(pyrimidyl-2yloxy)benzaldehyde (20 mmol) and acetic acid (0.1 mL) were mixed in a 100 mL round bottomed flask and refuxed in methanol for 10 h. The solid was filtered and washed with methanol and the remaining solid was dried to give (4-tolyloxy)-pyrimidyl chitosan Schiff base (4-TPSCS), yellow powder; yield 5.0 g (72.8%). 13 C NMR (300 MHz, ı): 25 (Ac-C), 55–62 (C-2, C-6), 71–78 (C-3, C-5), 83 (C-4), 106 (C-1), 115–126 (Ph-C), 130–139 (pyrimidyl-C), 154–158 ( C N), 160–169 (pyrimidyl-C).
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
85
Fig. 1. FT-IR spectra of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS.
A mixture of 4-TPSCS (5 mmol) and dimethyl phosphite (10 mmol) in 30 mL of toluene was stirred at 80 ◦ C for 10 h. After the reaction was complete, the residue was filtered, washed and dried to obtain (4-tolyloxy)-pyrimidyl-dimethyl-␣-amino-phosphonate chitosan (␣-ATPMCS), yield 1.89 g (83.6%), DS: 0.56. 31 P NMR (300 MHz, ı): 8.9 ( NH PC). 13 C NMR (300 MHz, ı): 25 (Ac-C), 35–45 (CH P), 55–60 (O CH3 ), 54–62 (C-2, C-6), 72–79 (C-3, C5), 83 (C-4), 106 (C-1), 116–125 (Ph-C), 130–140 (pyrimidyl-C), 158–167 (pyrimidyl-C). Similarly, (4-tolyloxy)-pyrimidyl-diethyl␣-aminophosphonate chitosan (␣-ATPECS) was prepared in the same way (Scheme 1), yield 1.60 g (62.5%), DS: 0.65. 31 P NMR (300 MHz, ı): 9.6 ( NH PC). 13 C NMR (300 MHz, ı): 16–20 (CH2 CH3 ), 25 (Ac-C), 35–46 (CH P), 56–61 (O CH2 CH3 ), 55–62 (C-2, C-6), 72–79 (C-3, C-5), 83 (C-4), 106 (C-1), 115–124 (Ph-C), 131–141 (pyrimidyl-C), 155–163 (pyrimidyl-C). 2.6. Antifungal assay Antifungal assay was evaluated against S. solani, R. solani, P. asparagi, and F. oxysporum in vitro by mycelium growth rate test [20]. The tested concentration was 250 mg/L, 500 mg/L, and 1000 mg/L, respectively. Petri dishes (9 cm diameter) containing 15 mL of potato dextrose agar (PDA) were used for antifungal activity assay. An agar plug of each fungal inoculum, removed from previous culture of each fungal pathogen, was placed upside down in the center of each Petri dish. The mixed medium without sample was used as the blank control. When the mycelium of fungi reached the edges of the blank control plate, the antifungal index was calculated with the following equation: Antifungal index (%) =
Db − Dt × 100 Db
Here, Dt is hyphal diameter in the test plate and Db is hyphal diameter in the blank control.
Triplicate of each test were carried out and the results were averaged. Results with P < 0.05 were considered statistically significant. 2.7. Initial antifungal mode of action studies The initial antifungal mechanism of the derivatives was studied in F. oxysporum model. 2.7.1. SEM observations of mycelia F. oxysporum was incubated for a week and then SEM analysis was employed to examine the structural changes of fungal samples at 100 mg/L dose. 2.7.2. Effect of ˛-ATPMCS and ˛-ATPECS on the relative permeability rate of cell membrane The mycelial of F. oxysporum which was incubated in potato dextrose (PD) for a week, and then polyoxin, ␣-ATPMCS and ␣-ATPECS were added to the PD to obtain a concentration of 100 mg/L, respectively. The conductivities at 0 (J0 ), 10, 20, 30, 60, 120 min were measured, gradually. In the end, the relative conductivity value was calculated: J = J − J0 . 2.7.3. Detection of mycelial pyruvate content Mycelial pyruvate content was determined according to the method described by Liu and Huang [21]. The upper clear layer (0.2 mL) of mycelial extract and distilled water (2.8 mL) were mixed with 2,4-dinitrophenylhydrazine (1.0 mL, 1 mg/mL), and then the mixture was swayed evenly for 10 min at 37 ◦ C. Next, sodium hydroxide (5.0 mL, 1.5 mol/L) was added and swayed uniformly. The absorbance value of the mixture measured at 520 nm was converted into the value of pyruvate content (mg/L) by the standard curve of pyruvate. Distilled water served as the control.
86
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Fig. 2. Solid state CP-MAS 13 C NMR spectra of CS, ␣-ATPMCS and ␣-ATPECS (a); 31 P NMR spectra of ␣-ATPMCS and ␣-ATPECS obtained with a single pulse experiment (b).
3. Results and discussion 3.1. Preparation and characterization of (4-tolyloxy)-pyrimidyl-˛-aminophosphonates chitosan ␣-Aminophosphonates are an important class of compounds with diverse biological activities such as antiviral, antifungal properties. Among methods for synthesis of ␣-aminophosphonates, the nucleophilic addition of dialkylphosphites to imines is thought to be the most useful method. Thus, (4-tolyloxy)pyrimidyl-␣-aminophosphonates chitosan was prepared via reaction of (4-tolyloxy)-pyrimidyl chitosan Schiff base (4-TPSCS) with dialkylphosphites described in Scheme 1. Their structures were well defined by FT-IR, 13 C NMR, 31 P NMR, elemental analysis, XRD, DSC, and SEM. Fig. 1 showed FT-IR spectra of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS. The broad band ranged from 3300 cm−1 to 3440 cm−1 attributed to OH and NH stretching vibration. The peak at 1596 cm−1 was for NH2 . And the peaks at 1152 cm−1 , 1087 cm−1 and 1031 cm−1 assigned to asymmetric stretching of the C O C. Compared with CS, 4-TPSCS showed new absorption peaks appear at 1648 cm−1 and 1581 cm−1 corresponding to the C N vibrations characteristic of imines and C C stretching vibrations. There was also a new strong peak observed at 1394 cm−1 ( C N).
Additionally, the peak at 1596 cm−1 of the NH2 disappeared. All these meant amino has been substituted and 4-TPSCS was obtained. For ␣-ATPMCS, new peaks at 1403 cm−1 ( C N), 1151 cm−1 ( P O), 1072 cm−1 ( P O C), 997 cm−1 ( P C) were observed. Hence, it was indicated that ␣-ATPMCS had been successfully obtained. Similar to ␣-ATPMCS, new peaks for ␣-ATPECS at 1386 cm−1 ( C N), 1162 cm−1 ( P O), 1074 cm−1 ( P O C), 986 cm−1 ( P C) confirmed structure of ␣-ATPECS [17]. The structures of ␣-ATPMCS and ␣-ATPECS were also confirmed by 13 C NMR and 31 P NMR (Fig. 2). The 13 C NMR spectrum of chitosan and its derivatives were shown in Fig. 2(a). Their signals can be well identified and were listed in Section 2 [22,23]. Compared with chitosan, new peaks for 4-TPSCS at 154–158 ppm attributed to C N (Schiff base group), 115–126 ppm (Ph-C), 130–139 ppm (pyrimidylC), and 160–169 ppm (pyrimidyl-C) demonstrated its structure. For ␣-ATPMCS, the signal relative to C N (Schiff base group) became weak and new peaks at 35–45 ppm (CH P), 55–60 ppm (O CH3 ), 116–125 ppm (Ph-C), 130–140 ppm (pyrimidyl-C), and 158–167 ppm (pyrimidyl-C) were observed. These results indicated ␣-ATPMCS has been obtained. Similarly, ı = 16–20 ppm (CH2 CH3 ); ı = 37–46 ppm (CH P); 56–61 ppm (O CH2 CH3 ), ı = 115–124 ppm (Ph C), ı = 131–141 ppm (pyrimidyl-C), and ı = 155–163 ppm (pyrimidyl-C) confirmed the structures of ␣-ATPECS. Structures of the obtained chitosan derivatives were further verified via 31 P
Fig. 3. XRD patterns of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS.
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
87
Fig. 4. DSC curves of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS.
NMR spectra. The observed signal at 8.9 ppm (phosphonate-P) for ␣-ATPMCS and 9.6 ppm (phosphonate-P) for ␣-ATPECS indicated the presence of phosphorus (Fig. 2(b)). This result also agreed with literature reported before [24]. The XRD spectra of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS were shown in Fig. 3. There were two typical reflections at 10.9◦ and 20.2◦ for chitosan. The reflection at 10.9◦ was assigned to crystal form I and the strongest peak appeared at 20.2◦ attributed to crystal form II [25]. For 4-TPSCS, the peaks slightly shifted to 10.5◦ and 19.9◦ , respectively. And there was a new sharp peak appeared at 5.3◦ . It indicated the crystalline structure of chitosan changed via
chemical modification. The crystalline structure transformed from amorphous structure to a relatively crystalline in CS to 4-TPSCS. Compared with p-TPSCS, the peak at 5.3◦ and 10.5◦ disappeared and the reflection at 19.9◦ became weak and smooth. X-ray diffraction exposed the differences among CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS. The chemical modification of chitosan changed the elemental composition of the polymer and the macromolecular conformation was ruptured, which was consistent with the results mentioned above. Polysaccharides are normally found to have a high hydrophilic capacity. The thermodynamics properties relate to their primary
Fig. 5. Surface morphology of CS (a), (4-TPSCS) (b), ␣-ATPMCS (c) and ␣-ATPECS (d).
88
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Table 1 Antifungal activitya of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS against R. solani, F. oxysporum, P. asparagi, and S. solani. Sample
Concentration (mg/L)
R. solani
CS
250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000
9.9 ± 1.4 24.5 ± 1.3 38.8 ± 4.2 47.1 ± 2.2 66.1 ± 0.1 83.7 ± 1.3 46.5 ± 4.1 67.2 ± 5.8 83.5 ± 2.2 0 15.1 ± 2.9 25.4 ± 0.5 55.4 ± 1.6 69.7 ± 1.3 87.3 ± 1.7
␣-ATPMCS
␣-ATPECS
4-TPSCS
Polyoxin
a
F. oxysporum 7.9 ± 1.3 54.9 ± 2.3 83.9 ± 2.0 100.0 100.0 100.0 100.0 100.0 100.0 0 13.3 ± 4.0 48.9 ± 3.0 32.1 ± 4.2 55.6 ± 3.5 65.7 ± 4.0
P. asparagi
S. solani
27.5 ± 3.6 46.1 ± 2.2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 10.3 ± 2.2 23.0 ± 3.4 58.1 ± 4.7 37.2 ± 2.2 56.6 ± 2.6 61.9 ± 2.2
12.3 ± 2.0 41.2 ± 1.7 50.9 ± 2.9 34.5 ± 1.7 47.9 ± 5.0 68.2 ± 2.9 30.6 ± 2.9 54.7 ± 1.7 100.0 0 8.5 ± 1.0 39.3 ± 2.9 100.0 100.0 100.0
Values are given as mean (SD) of three experiments.
Fig. 6. Inhibitory effect of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS on F. oxysporum (a) and P. asparagi (b). Error bars are standard deviations (SD) of 3 measurements.
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
89
Fig. 7. Ultrastructure of mycelium of F. oxysporum treated at 100 mg/L by ␣-ATPMCS (a), ␣-ATPECS (b), polyoxin (c), and blank control (d).
and super molecular structures [26]. Therefore, the endothermic peak was expected to reflect physical and molecular changes during chemical modification. Thermal properties of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS were evaluated by DSC (Fig. 4). Compared with CS and 4-TPSCS, a new strong sharp endothermic peak attributed to thermal decomposition of ␣-ATPMCS and ␣-ATPECS appeared around 200 ◦ C, 220 ◦ C, respectively. It was inferred the thermal properties of chitosan decreased via chemical modification, the crystal structure of chitosan changed as a consequence of the formation of a chitosan derivative [27]. It was shown CS and 4-TPSCS has a compact and flat morphology, while ␣-ATPMCS and ␣-ATPECS exhibited loose and porous structures (Fig. 5). These were in accordance with the XRD results.
It was deduced the improvement of water-solubility may result from the change of dense structure for CS and 4-TPSCS to porous structure for ␣-ATPMCS and ␣-ATPECS. 3.2. Antifungal activity of (4-tolyloxy)-pyrimidyl-˛-aminophosphonates chitosan In this study, inhibitory effect of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS on phytopatho-genic fungi was studied. The fungi used in the bioassay, S. solani, R. solani, P. asparagi, and F. oxysporum, were collected and isolated from corresponding crops. The results of preliminary bioassays were compared with that of a commercial fungicide polyoxin.
Fig. 8. Effect of ␣-ATPMCS and ␣-ATPECS on change of medium electrical conductivity of F. oxysporum.
90
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
As shown in Table 1, both ␣-ATPMCS and ␣-ATPECS exhibited potent antifungal activity against all the tested fungi. As main observations, it can be stated that both ␣-ATPMCS and ␣-ATPECS exhibited significant inhibitory effect on the fungi especially P. asparagi and F. oxysporum. However, significant lowering of activity for 4-TPSCS was noticed. It was inferred introduction pyrimidines moiety into chitosan backbone do not necessarily imply the enhancement of activity. Our previous study also showed that Schiff base chitosan derivatives had weaker antifungal activity than chitosan. For example, Guo et al. reported the Schiff bases of chitosan (As, Bs) have a slight activity against B. cinerea Pers. The inhibitory indices of them at 1000 mg/L were 26.8% and 33.5%, while the inhibitory index of chitosan was 45.4% [28]. It could be seen that both ␣-ATPMCS and ␣-ATPECS, exhibiting promising antifungal activities even better than that of the fungicide polyoxin. At the concentration of 250 mg/L, ␣-ATPMCS and ␣-ATPECS inhibited growth of P. asparagi, and F. oxysporum at 100%, which are obviously better than that of polyoxin (32.1% against P. asparagi, and 37.2% against F. oxysporum at 250 mg/L). However, polyoxin was more active in controlling S. solani (100% against S. solani) than ␣-ATPMCS (34.5%) and ␣-TPECS (30.6%) at 250 mg/L. For R. solani, it was almost equally sensitive to polyoxin (55.4%), ␣-ATPMCS (47.1%) and ␣-ATPECS (46.5%). It is very interesting to note that ␣-ATPMCS and ␣-ATPECS still showed good activities against P. asparagi, and F. oxysporum at lower concentrations (Fig. 6). F. oxysporum was the most sensitive fungus among the tested fungi to ␣-ATPMCS and ␣-ATPECS. At 31.25 mg/L, the inhibitory index of ␣-ATPMCS and ␣-ATPECS against F. oxysporum was 27.5% and 24.8%, much stronger than that of polyoxin (8.5%). From the above bioassay results, we found that (4-tolyloxy)pyrimidyl-␣-aminophosphonates chitosan derivatives obvious inhibited the selected fungi compared with chitosan. Both ␣ATPMCS and ␣-ATPECS had broad-spectrum antifungal activity. 3.3. Initial mechanism of action studies The cytoplasm and membrane of micro-organisms is the target for many inhibition agents [29–31]. When the membranes become compromised by interaction with antimicrobial agents, low molecular mass species such as K+ and PO4 3− leach out and then followed by DNA, RNA and other materials. Although the actual antimicrobial mechanism of chitosan has not yet fully understood, but it has been suggested to involve with breakdown of the membrane barrier [32–36]. So in this study, to gain insight into the mode of action of the chitosan derivatives, ␣-ATPMCS and ␣-ATPECS were tested for their capacity of affecting membrane permeability of F. oxysporum. Their effect on the relative permeability rate of cell membrane and mycelial pyruvate content was determined. It was shown the images of mycelia obtained from the edge of F. oxysporum culture in the blank control (untreated samples), demonstrating hyphae with typical smooth surface (Fig. 7). While samples treated with ␣-ATPMCS and ␣-ATPECS, the hyphae lost their smoothness and formed abnormal bulges and branches on the surface of fungal hyphae, indicating that inhibited the growth of F. oxysporum by deforming the structure of fungal hyphae. SEM observations of mycelia from the colonies displayed branched conidia with round and chain-like structures. While treated with ␣-ATPMCS and ␣-ATPECS, germination of F. oxysporum conidia was partly inhibited and conidial development of F. oxysporum was suppressed. The results suggest that ␣-ATPMCS and ␣-ATPECS can distort and damage the conidia of F. oxysporum. The effect of ␣-ATPMCS and ␣-ATPECS on membrane permeability was detected in F. oxysporum at 100 mg/L, respectively. When the treatment time was lower than 20 min, relative conductivity with ␣-ATPMCS and ␣-ATPECS treatment was found to be lower
Fig. 9. Change of pyruvic acid of F. oxysporum treated with ␣-ATPMCS and ␣-ATPECS.
than that treated with polyoxin. The results may be attributed to the fact that polyoxin as a small molecule destroyed the cell membrane more quickly than ␣-ATPMCS and ␣-ATPECS. When the treatment time was longer than 20 min, the relative conductivity with ␣ATPECS treatment was higher than that of polyoxin (Fig. 8). It is inferred that this kind of chitosan derivatives may interact with the membrane and cause the leach out of low molecular nucleic acid, proteins, and so on. Fig. 9 showed the change of pyruvic acid of F. oxysporum treated with ␣-ATPMCS and ␣-ATPECS. It was observed that pyruvic acid of F. oxysporum treated with the samples are all lower than the blank control. It may be attributed to their inhibitory effect on the activity of lactate dehydrogenase, thereby inducing the decrease of the pyruvate content [37,38]. Additionally, pyruvic acid of F. oxysporum treated with ␣-ATPMCS and ␣ATPECS was lower than polyxin treated, which was in agreement with the antifungal results. Mycelial pyruvate plays an important role in energy metabolism of carbon source pathway. In this way, it was indicated the chitosan derivatives interfere with the construction of cell architecture and caused hypha growth inhibition. On the basis of the above results, it was concluded that ␣-ATPMCS and ␣-ATPECS executed antifungal properties may associated with energy metabolism and cell wall-degrading. 4. Conclusions In an attempt to discover novel antifungal structures chitosan derivatives with significant activities against crop-threatening fungi, a series of (4-tolyloxy)-pyrimidyl-␣-aminophosphonates chitosan derivatives were prepared. Their structures were confirmed byFT-IR, 13 C NMR, 31 P NMR, elemental analysis, XRD, DSC, and SEM. Antifungal activity of them against S. solani, R. solani, P. asparagi, and F. oxysporum was evaluated. In general, ␣-ATPMCS and ␣-ATPECS obvious inhibited the tested fungi compared with chitosan. And both of them had broad-spectrum antifungal activity. P. asparagi and F. oxysporum were more sensitive to the chitosan derivatives than S. solani and R. solani. According with the preliminary results in these assays, ␣-ATPMCS and ␣-ATPECS even showed much stronger inhibitory effects on P. asparagi and F. oxysporum than the commercial fungicide polyoxin. Additionally, initial mechanism of action studies exhibited ␣-ATPMCS and ␣-ATPECS may affect membrane permeability of F. oxysporum and inhibit mycelial pyruvate content of the fungus. It is important to notice that the derivatives might be good candidates for further design of antifungal agents.
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Acknowledgements The study was supported by the Public Science and Technology Research Funds Projects of Ocean (No. 201305016-2), the Science and Technology Development Program of Shandong Province (No. 2012GHY11530), the National High Technology Research and Development Program (“863” Program) of China (2011AA09070405), and the Innovational Foundation of Chinese Academy of Sciences (KZCX2-EW-Q214). References [1] R.A.A. Muzzarelli, J. Boudrant, D. Meyer, N. Manno, M. DeMarchis, M.G. Paoletti, Carbohydr. Polym. 87 (2012) 995–1012. [2] M.N.V.R. Kumar, React. Funct. Polym. 46 (2000) 1–27. [3] T.S. Anirudhan, S. Sandeep, Polym. Chem. UK 2 (2011) 2052–2061. [4] R.S. Jagadish, K.N. Divyashree, P. Viswanath, P. Srinivas, B. Raj, Carbohydr. Polym. 87 (2012) 110–116. [5] S.L. Kosaraju, R. Weerakkody, M.A. Augustin, J. Agric. Food Chem. 58 (2010) 12449–12455. [6] E.I. Rabea, M.E.-T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Biomacromolecules 4 (2003) 1457–1465. ˜ [7] A.N.H.-L.S. Bautista-Banos, M.G. Velázquez-del Valle, M. Hernández-López, E. Ait Barka, E. Bosquez-Molina, C.L. Wilson, Crop Prot. 25 (2006) 108–118. [8] C.K.S. Pillai, W. Paul, C.P. Sharma, Prog. Polym. Sci. 34 (2009) 641–678. [9] K.V. Harish Prashanth, R.N. Tharanathan, Trend Food Sci. Technol. 18 (2007) 117–131. [10] M.N.V.R. Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A.J. Domb, Chem. Rev. 104 (2004) 6017–6084. [11] V.K. Mourya, N.N. Inamdar, React. Funct. Polym. 68 (2008) 1013–1051. [12] R. Belalia, S. Grelier, M. Benaissa, V. Coma, J. Agric. Food Chem. 56 (2008) 1582–1588. [13] I. Sebti, E. Chollet, P. Degraeve, C. Noel, E. Peyrol, J. Agric. Food Chem. 55 (2007) 693–699.
91
[14] S. De Lombaert, L.B. Stamford, L. Blanchard, J. Tan, D. Hoyer, C.G. Diefenbacher, D. Wei, E.M. Wallace, M.A. Moskal, P. Savage, A.Y. Jeng, Bioorg. Med. Chem. Lett. 7 (1997) 1059–1064. [15] A. Mucha, P. Kafarski, Ł. Berlicki, J. Med. Chem. 54 (2011) 5955–5980. [16] G. Forlani, A. Occhipinti, Ł. Berlicki, G. Dziedzioła, A. Wieczorek, P. Kafarski, J. Agric. Food Chem. 56 (2008) 3193–3199. [17] Z. Zhong, P. Li, R. Xing, X. Chen, S. Liu, Int. J. Biol. Macromol. 45 (2009) 255–259. [18] R.J. Milling, C.J. Richardson, Pestic. Sci. 45 (1995) 43–48. [19] D. Errampalli, N.R. Brubacher, Biol. Control 36 (2006) 49–56. [20] Y. Qin, S. Liu, R. Xing, H. Yu, K. Li, X. Meng, R. Li, P. Li, Carbohydr. Polym. 89 (2012) 388–393. [21] F. Liu, Y. Huang, Pestic. Biochem. Phys. 101 (2011) 248–255. [22] H. Saito, R. Tabeta, K. Ogawa, Macromolecules 20 (1987) 2424–2430. [23] C. Lefay, Y. Guillaneuf, G. Moreira, J.J. Thevarajah, P. Castignolles, F. Ziarelli, E. Bloch, M. Major, L. Charles, M. Gaborieau, D. Bertin, D. Gigmes, Polym. Chem. UK 4 (2013) 322–328. [24] R. Zeng, H. Fu, Y. Zhao, Macromol. Rapid Commun. 27 (2006) 548–552. [25] Y. Wu, Y. Zheng, W. Yang, C. Wang, J. Hu, S. Fu, Carbohydr. Polym. 59 (2005) 165–171. [26] G.O. Phillips, S. Takigami, M. Takigami, Food Hydrocolloids 10 (1996) 11–19. [27] Y. Qin, R. Xing, S. Liu, K. Li, X. Meng, R. Li, J. Cui, B. Li, P. Li, Carbohydr. Polym. 87 (2012) 2664–2670. [28] Z. Guo, R. Xing, S. Liu, Z. Zhong, X. Ji, L. Wang, P. Li, Carbohyd. Res. 342 (2007) 1329–1332. [29] Y. Shai, Pept. Sci. 66 (2002) 236–248. [30] I. Kubo, K.i. Fujita, S.H. Lee, J. Agric. Food Chem. 49 (2001) 1607–1611. [31] B. Hwang, J.-S. Hwang, J. Lee, D.G. Lee, Biochem. Biophys. Res. Commun. 400 (2010) 352–357. [32] X. Li, X. Feng, S. Yang, G. Fu, T. Wang, Z. Su, Carbohydr. Polym. 79 (2010) 493–499. [33] I.M. Helander, E.L. Nurmiaho-Lassila, R. Ahvenainen, J. Rhoades, S. Roller, Int. J. Food Microbiol. 71 (2001) 235–244. [34] M. Kong, X.G. Chen, K. Xing, H.J. Park, Int. J. Food Microbiol. 144 (2010) 51–63. [35] A. El Ghaouth, J. Arul, A. Asselin, N. Benhamou, Mycol. Res. 96 (1992) 769–779. [36] J. Je, S. Kim, J. Agric. Food Chem. 54 (2006) 6629–6633. [37] F. Liu, X. Luo, B. Song, P. Bhadury, S. Yang, L. Jin, W. Xue, D. Hu, Bioorg. Med. Chem. 16 (2008) 3632–3640. [38] W. Xu, S. Yang, P. Bhadury, J. He, M. He, L. Gao, D. Hu, B. Song, Pestic. Biochem. Phys. 101 (2011) 6–15.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis and antifungal properties of (4-tolyloxy)-pyrimidyl-␣-aminophosphonates chitosan derivatives Yukun Qin a , Ronge Xing a , Song Liu a , Huahua Yu a , Kecheng Li a , Linfeng Hu a,b , Pengcheng Li a,∗ a b
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China School of Chemistry & Chemical Engineering, Henan Institute of Science and Technology, Xinxiang 453003, China
a r t i c l e
i n f o
Article history: Received 12 September 2013 Received in revised form 17 October 2013 Accepted 21 October 2013 Available online xxx Keywords: ␣-Aminophosphonate chitosan Synthesis Antifungal activity
a b s t r a c t A novel class of ␣-aminophosphonate chitosan derivatives was investigated. These chitosan derivatives consist of (4-tolyloxy)-pyrimidyl-dimethyl-␣-amino-phosphonate chitosan (␣-ATPMCS) and (4-tolyloxy)-pyrimidyl-diethyl-␣-aminophosphonate chitosan (␣-ATPECS). Their structures were well defined. Antifungal activity of them against some crop-threatening pathogenic fungi was tested in vitro. The derivatives were found to have a broad-spectrum antifungal activity that was obviously enhanced compared with chitosan. At 250 mg/L, both ␣-ATPMCS and ␣-ATPECS even inhibited growth of Phomopsis asparagi (Sacc.) (P. asparagi) and Fusarium oxysporum (F. oxysporum) at 100%, which was even stronger than polyoxin whose antifungal index was 37.2% and 32.1%, respectively. Additionally, the initial mechanism of the chitosan derivatives in F. oxysporum model was studied. It was found that the derivatives may have an effect on membrane permeability of the fungi. The results demonstrated the derivatives may serve as attractive candidates in crop protection. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitosan (poly--(1,4)-d-glucosamine) is an abundant biopolymer obtained from chitin. As an important renewable biopolymer, it attracts much attention in such fields as pharmacology, agriculture, materials, and food due to its good biocompatibility, biodegradability, and non-toxicity [1–5]. Antimicrobial activities of chitosan have been widely explored in vitro and in vivo. It was proved that chitosan can control a variety of bacteria and fungi [6,7]. However, chitosan is generally insoluble in water and most organic solvents for its strongly hydrogenbonding network structure [8]. Additionally, its antimicrobial activity is relatively lower than that of commercial antimicrobial agents. Thus, the use of chitosan as an antimicrobial agent has been restricted. Combinations of high molar mass polymers and low molar active molecules normally can take synergistic advantage of their unique properties, including the mechanical properties of polymers and bioactive properties of the small molecules. Thus, chemical modifications are an efficient approach in improving both water solubility and antimicrobial activity of chitosan [9–11]. Many
∗ Corresponding author at: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. Tel.: +86 532 82898707; fax: +86 532 82968951. E-mail addresses: [email protected] (S. Liu), [email protected], [email protected] (P. Li). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.023
attempts have been made to create new chitosan derivatives with an enhanced solubility and antimicrobial activity [12,13]. These derivatives include alkylation, acylation, quaternization, thiosemicarbazones chitosan and so on. ␣-Aminophosphonates, a kind of natural amino acid analogs, is an important class of compounds acted such as enzyme inhibitors [14], pharmacological agents [15], and pesticides [16]. In previous study, we reported the synthesis of 2-(␣-arylaminophosphonate)-chitosan via introduction ␣aminophosphonates group into chitosan backbone [17]. The obtained ␣-aminophosphonates chitosan showed a significant inhibiting effect on the investigated fungi compared with origin chitosan. However, their structures were not fully characterized (only in FT-IR) and the antifungal results were also not so satisfactory for them to be served as fungicidal candidates. Therefore, it was necessary to discover more effective derivatives of this kind via further chemical optimization. Heterocyclic compounds particularly six-membered compounds have occupied a prominent place among various classes of organic compounds for their diverse biological activities. Among a wide variety of heterocycles, pyrimidines are the integral part of nucleic acids and have been associated with a number of biological activities. They have played an important role in pesticide chemistry [18,19]. Fungicides with pyrimidines such as cyprodinil, ferimzone, mepanipyrim are widely used in crop protection. On the basis of the literature, we made the further assumption that combination of the pyrimidines
84
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Scheme 1. Synthesis of 4-(pyrimidyl-2-yloxy)benzaldehyde (a) and (4-tolyloxy)-pyrimidyl-␣-aminophosphonates chitosan (b).
moiety with ␣-aminophosphonates chitosan may enhance their antifungal activity. In this study, with the aim to develop more effective ␣-aminophosphonates chitosan derivatives, we focused our attention on grafting fungicidal pyrimidyl molecules that are known to inhibit crop-threatening pathogenic fungi on ␣aminophosphonates chitosan framework. To validate the hypothesis that a pyrimidyl moiety is required for improving the antifungal activity, the inhibitory effects of new obtained chitosan derivatives were tested in vitro. 2. Experimental 2.1. Materials Chitosan, with 87% degree of deacetylation and averagemolecular weight of 230 kDa, was supplied from Qingdao Baicheng Biochemical Corp. Polyoxin, 10% wettable powders, was purchased from Kaken Pharmaceutical Co., Ltd. 2-Chloropyrimidine was purchased from Chengdu Best Reagent Co., Ltd. (China). 4-Hydroxybenzaldehyde was purchased from Shanghai Shiyi Chemical Reagent Co., Ltd. (China). Other commercial chemical reagents used in the experiment were obtained from Sinopharm Group Chemical Reagent Co. Ltd. All of the regents were analytical grade and used without further purification. 2.2. Fungal strains The crop pathogenic fungi tested were obtained from Qingdao Academy of Agricultural Sciences. There were a total of four fungi strains: Rhizoctonia solani Kühn (R. solany), Phomopsis asparagi (Sacc.) (P. asparagi), and Fusarium oxysporum (F. oxysporum). Cultures of each of the fungi were maintained on potato dextrose agar (PDA) medium and were stored at 4 ◦ C. 2.3. Characterization Fourier transform infrared (FTIR) spectrum was measured in the 4000–400 cm−1 . Solid-state 13 C CP/MAS NMR spectra were performed on a Bruker AC-300 spectrometer. A ramped contact-power cross-polarization (CP) pulse sequence was applied and a doubleair-bearing Magic Angle Spinning (MAS) probe with 7 mm external diameter sample rotors was employed. Time domain size is 2048. The number of scans is 1000. 31 P NMR was recorded on samples
with a single pulse sequence model used a Bruker AC-300 spectrometer. The elemental analysis (C, H, and N) was carried out using a Vario EL-III elemental analyzer. The percentages of carbon, hydrogen and nitrogen were estimated and the degree of substitution (DS) of the chitosan derivatives was calculated on the basis of the percentage. X-ray diffraction (XRD) measurement of the powder samples were performed with a D8 Advance diffractometer (Bruker) with Cu target ( = 0.154 nm) at 40 kV and the scanning scope of 2 was 5–50◦ . Differential scanning calorimetry (DSC) was performed using the Pyris Diamond DSC (Perkin Elmer). The samples were heated from 50 ◦ C to 230 ◦ C under 10 ◦ C/min. Nitrogen gas was used in the experiment to confirm thermal behavior. The surface morphology of the samples was analyzed by scanning electron microscopy by using KYKY-2800B SEM. 2.4. Synthesis of 4-(pyrimidyl-2-yloxy)benzaldehyde A mixture of 4-hydroxybenzaldehyde (50 mmol, 6.1 g) and potassium carbonate (60 mmol, 8.28 g) was stirred in a 250 mL round bottomed flask with 100 mL DMF for half an hour at room temperature. Then 2-chloropyrimidine (50 mmol, 6.1 g) was added and the reaction mixture was stirred for another 10 h at 100 ◦ C until the 2-chloropyrimidine disappeared (TLC control). The residue was dissolved in ethyl acetate and washed with water. The combined organic layer was dried over MgSO4 and concentrated in vacuo to give the crude yellow oil. The residue was purified by column chromatography on silica gel and the product was obtained with 90.0% yield. 1 H NMR (600 MHz, DMSO-d6 , ı): 10.00 (s, 1H, CHO), 8.65 (d, 2H, pyrimidyl-H), 7.97 (d, 2H, Ph-H), 7.33 (d, 2H, Ph-H), 7.32 (t, 1H, pyrimidyl-H). 2.5. Synthesis of (4-tolyloxy)-pyrimidyl-˛-aminophosphonates chitosan Chitosan (20 mmol), the corresponding 4-(pyrimidyl-2yloxy)benzaldehyde (20 mmol) and acetic acid (0.1 mL) were mixed in a 100 mL round bottomed flask and refuxed in methanol for 10 h. The solid was filtered and washed with methanol and the remaining solid was dried to give (4-tolyloxy)-pyrimidyl chitosan Schiff base (4-TPSCS), yellow powder; yield 5.0 g (72.8%). 13 C NMR (300 MHz, ı): 25 (Ac-C), 55–62 (C-2, C-6), 71–78 (C-3, C-5), 83 (C-4), 106 (C-1), 115–126 (Ph-C), 130–139 (pyrimidyl-C), 154–158 ( C N), 160–169 (pyrimidyl-C).
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
85
Fig. 1. FT-IR spectra of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS.
A mixture of 4-TPSCS (5 mmol) and dimethyl phosphite (10 mmol) in 30 mL of toluene was stirred at 80 ◦ C for 10 h. After the reaction was complete, the residue was filtered, washed and dried to obtain (4-tolyloxy)-pyrimidyl-dimethyl-␣-amino-phosphonate chitosan (␣-ATPMCS), yield 1.89 g (83.6%), DS: 0.56. 31 P NMR (300 MHz, ı): 8.9 ( NH PC). 13 C NMR (300 MHz, ı): 25 (Ac-C), 35–45 (CH P), 55–60 (O CH3 ), 54–62 (C-2, C-6), 72–79 (C-3, C5), 83 (C-4), 106 (C-1), 116–125 (Ph-C), 130–140 (pyrimidyl-C), 158–167 (pyrimidyl-C). Similarly, (4-tolyloxy)-pyrimidyl-diethyl␣-aminophosphonate chitosan (␣-ATPECS) was prepared in the same way (Scheme 1), yield 1.60 g (62.5%), DS: 0.65. 31 P NMR (300 MHz, ı): 9.6 ( NH PC). 13 C NMR (300 MHz, ı): 16–20 (CH2 CH3 ), 25 (Ac-C), 35–46 (CH P), 56–61 (O CH2 CH3 ), 55–62 (C-2, C-6), 72–79 (C-3, C-5), 83 (C-4), 106 (C-1), 115–124 (Ph-C), 131–141 (pyrimidyl-C), 155–163 (pyrimidyl-C). 2.6. Antifungal assay Antifungal assay was evaluated against S. solani, R. solani, P. asparagi, and F. oxysporum in vitro by mycelium growth rate test [20]. The tested concentration was 250 mg/L, 500 mg/L, and 1000 mg/L, respectively. Petri dishes (9 cm diameter) containing 15 mL of potato dextrose agar (PDA) were used for antifungal activity assay. An agar plug of each fungal inoculum, removed from previous culture of each fungal pathogen, was placed upside down in the center of each Petri dish. The mixed medium without sample was used as the blank control. When the mycelium of fungi reached the edges of the blank control plate, the antifungal index was calculated with the following equation: Antifungal index (%) =
Db − Dt × 100 Db
Here, Dt is hyphal diameter in the test plate and Db is hyphal diameter in the blank control.
Triplicate of each test were carried out and the results were averaged. Results with P < 0.05 were considered statistically significant. 2.7. Initial antifungal mode of action studies The initial antifungal mechanism of the derivatives was studied in F. oxysporum model. 2.7.1. SEM observations of mycelia F. oxysporum was incubated for a week and then SEM analysis was employed to examine the structural changes of fungal samples at 100 mg/L dose. 2.7.2. Effect of ˛-ATPMCS and ˛-ATPECS on the relative permeability rate of cell membrane The mycelial of F. oxysporum which was incubated in potato dextrose (PD) for a week, and then polyoxin, ␣-ATPMCS and ␣-ATPECS were added to the PD to obtain a concentration of 100 mg/L, respectively. The conductivities at 0 (J0 ), 10, 20, 30, 60, 120 min were measured, gradually. In the end, the relative conductivity value was calculated: J = J − J0 . 2.7.3. Detection of mycelial pyruvate content Mycelial pyruvate content was determined according to the method described by Liu and Huang [21]. The upper clear layer (0.2 mL) of mycelial extract and distilled water (2.8 mL) were mixed with 2,4-dinitrophenylhydrazine (1.0 mL, 1 mg/mL), and then the mixture was swayed evenly for 10 min at 37 ◦ C. Next, sodium hydroxide (5.0 mL, 1.5 mol/L) was added and swayed uniformly. The absorbance value of the mixture measured at 520 nm was converted into the value of pyruvate content (mg/L) by the standard curve of pyruvate. Distilled water served as the control.
86
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Fig. 2. Solid state CP-MAS 13 C NMR spectra of CS, ␣-ATPMCS and ␣-ATPECS (a); 31 P NMR spectra of ␣-ATPMCS and ␣-ATPECS obtained with a single pulse experiment (b).
3. Results and discussion 3.1. Preparation and characterization of (4-tolyloxy)-pyrimidyl-˛-aminophosphonates chitosan ␣-Aminophosphonates are an important class of compounds with diverse biological activities such as antiviral, antifungal properties. Among methods for synthesis of ␣-aminophosphonates, the nucleophilic addition of dialkylphosphites to imines is thought to be the most useful method. Thus, (4-tolyloxy)pyrimidyl-␣-aminophosphonates chitosan was prepared via reaction of (4-tolyloxy)-pyrimidyl chitosan Schiff base (4-TPSCS) with dialkylphosphites described in Scheme 1. Their structures were well defined by FT-IR, 13 C NMR, 31 P NMR, elemental analysis, XRD, DSC, and SEM. Fig. 1 showed FT-IR spectra of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS. The broad band ranged from 3300 cm−1 to 3440 cm−1 attributed to OH and NH stretching vibration. The peak at 1596 cm−1 was for NH2 . And the peaks at 1152 cm−1 , 1087 cm−1 and 1031 cm−1 assigned to asymmetric stretching of the C O C. Compared with CS, 4-TPSCS showed new absorption peaks appear at 1648 cm−1 and 1581 cm−1 corresponding to the C N vibrations characteristic of imines and C C stretching vibrations. There was also a new strong peak observed at 1394 cm−1 ( C N).
Additionally, the peak at 1596 cm−1 of the NH2 disappeared. All these meant amino has been substituted and 4-TPSCS was obtained. For ␣-ATPMCS, new peaks at 1403 cm−1 ( C N), 1151 cm−1 ( P O), 1072 cm−1 ( P O C), 997 cm−1 ( P C) were observed. Hence, it was indicated that ␣-ATPMCS had been successfully obtained. Similar to ␣-ATPMCS, new peaks for ␣-ATPECS at 1386 cm−1 ( C N), 1162 cm−1 ( P O), 1074 cm−1 ( P O C), 986 cm−1 ( P C) confirmed structure of ␣-ATPECS [17]. The structures of ␣-ATPMCS and ␣-ATPECS were also confirmed by 13 C NMR and 31 P NMR (Fig. 2). The 13 C NMR spectrum of chitosan and its derivatives were shown in Fig. 2(a). Their signals can be well identified and were listed in Section 2 [22,23]. Compared with chitosan, new peaks for 4-TPSCS at 154–158 ppm attributed to C N (Schiff base group), 115–126 ppm (Ph-C), 130–139 ppm (pyrimidylC), and 160–169 ppm (pyrimidyl-C) demonstrated its structure. For ␣-ATPMCS, the signal relative to C N (Schiff base group) became weak and new peaks at 35–45 ppm (CH P), 55–60 ppm (O CH3 ), 116–125 ppm (Ph-C), 130–140 ppm (pyrimidyl-C), and 158–167 ppm (pyrimidyl-C) were observed. These results indicated ␣-ATPMCS has been obtained. Similarly, ı = 16–20 ppm (CH2 CH3 ); ı = 37–46 ppm (CH P); 56–61 ppm (O CH2 CH3 ), ı = 115–124 ppm (Ph C), ı = 131–141 ppm (pyrimidyl-C), and ı = 155–163 ppm (pyrimidyl-C) confirmed the structures of ␣-ATPECS. Structures of the obtained chitosan derivatives were further verified via 31 P
Fig. 3. XRD patterns of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS.
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
87
Fig. 4. DSC curves of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS.
NMR spectra. The observed signal at 8.9 ppm (phosphonate-P) for ␣-ATPMCS and 9.6 ppm (phosphonate-P) for ␣-ATPECS indicated the presence of phosphorus (Fig. 2(b)). This result also agreed with literature reported before [24]. The XRD spectra of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS were shown in Fig. 3. There were two typical reflections at 10.9◦ and 20.2◦ for chitosan. The reflection at 10.9◦ was assigned to crystal form I and the strongest peak appeared at 20.2◦ attributed to crystal form II [25]. For 4-TPSCS, the peaks slightly shifted to 10.5◦ and 19.9◦ , respectively. And there was a new sharp peak appeared at 5.3◦ . It indicated the crystalline structure of chitosan changed via
chemical modification. The crystalline structure transformed from amorphous structure to a relatively crystalline in CS to 4-TPSCS. Compared with p-TPSCS, the peak at 5.3◦ and 10.5◦ disappeared and the reflection at 19.9◦ became weak and smooth. X-ray diffraction exposed the differences among CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS. The chemical modification of chitosan changed the elemental composition of the polymer and the macromolecular conformation was ruptured, which was consistent with the results mentioned above. Polysaccharides are normally found to have a high hydrophilic capacity. The thermodynamics properties relate to their primary
Fig. 5. Surface morphology of CS (a), (4-TPSCS) (b), ␣-ATPMCS (c) and ␣-ATPECS (d).
88
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Table 1 Antifungal activitya of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS against R. solani, F. oxysporum, P. asparagi, and S. solani. Sample
Concentration (mg/L)
R. solani
CS
250 500 1000 250 500 1000 250 500 1000 250 500 1000 250 500 1000
9.9 ± 1.4 24.5 ± 1.3 38.8 ± 4.2 47.1 ± 2.2 66.1 ± 0.1 83.7 ± 1.3 46.5 ± 4.1 67.2 ± 5.8 83.5 ± 2.2 0 15.1 ± 2.9 25.4 ± 0.5 55.4 ± 1.6 69.7 ± 1.3 87.3 ± 1.7
␣-ATPMCS
␣-ATPECS
4-TPSCS
Polyoxin
a
F. oxysporum 7.9 ± 1.3 54.9 ± 2.3 83.9 ± 2.0 100.0 100.0 100.0 100.0 100.0 100.0 0 13.3 ± 4.0 48.9 ± 3.0 32.1 ± 4.2 55.6 ± 3.5 65.7 ± 4.0
P. asparagi
S. solani
27.5 ± 3.6 46.1 ± 2.2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 10.3 ± 2.2 23.0 ± 3.4 58.1 ± 4.7 37.2 ± 2.2 56.6 ± 2.6 61.9 ± 2.2
12.3 ± 2.0 41.2 ± 1.7 50.9 ± 2.9 34.5 ± 1.7 47.9 ± 5.0 68.2 ± 2.9 30.6 ± 2.9 54.7 ± 1.7 100.0 0 8.5 ± 1.0 39.3 ± 2.9 100.0 100.0 100.0
Values are given as mean (SD) of three experiments.
Fig. 6. Inhibitory effect of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS on F. oxysporum (a) and P. asparagi (b). Error bars are standard deviations (SD) of 3 measurements.
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
89
Fig. 7. Ultrastructure of mycelium of F. oxysporum treated at 100 mg/L by ␣-ATPMCS (a), ␣-ATPECS (b), polyoxin (c), and blank control (d).
and super molecular structures [26]. Therefore, the endothermic peak was expected to reflect physical and molecular changes during chemical modification. Thermal properties of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS were evaluated by DSC (Fig. 4). Compared with CS and 4-TPSCS, a new strong sharp endothermic peak attributed to thermal decomposition of ␣-ATPMCS and ␣-ATPECS appeared around 200 ◦ C, 220 ◦ C, respectively. It was inferred the thermal properties of chitosan decreased via chemical modification, the crystal structure of chitosan changed as a consequence of the formation of a chitosan derivative [27]. It was shown CS and 4-TPSCS has a compact and flat morphology, while ␣-ATPMCS and ␣-ATPECS exhibited loose and porous structures (Fig. 5). These were in accordance with the XRD results.
It was deduced the improvement of water-solubility may result from the change of dense structure for CS and 4-TPSCS to porous structure for ␣-ATPMCS and ␣-ATPECS. 3.2. Antifungal activity of (4-tolyloxy)-pyrimidyl-˛-aminophosphonates chitosan In this study, inhibitory effect of CS, 4-TPSCS, ␣-ATPMCS and ␣-ATPECS on phytopatho-genic fungi was studied. The fungi used in the bioassay, S. solani, R. solani, P. asparagi, and F. oxysporum, were collected and isolated from corresponding crops. The results of preliminary bioassays were compared with that of a commercial fungicide polyoxin.
Fig. 8. Effect of ␣-ATPMCS and ␣-ATPECS on change of medium electrical conductivity of F. oxysporum.
90
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
As shown in Table 1, both ␣-ATPMCS and ␣-ATPECS exhibited potent antifungal activity against all the tested fungi. As main observations, it can be stated that both ␣-ATPMCS and ␣-ATPECS exhibited significant inhibitory effect on the fungi especially P. asparagi and F. oxysporum. However, significant lowering of activity for 4-TPSCS was noticed. It was inferred introduction pyrimidines moiety into chitosan backbone do not necessarily imply the enhancement of activity. Our previous study also showed that Schiff base chitosan derivatives had weaker antifungal activity than chitosan. For example, Guo et al. reported the Schiff bases of chitosan (As, Bs) have a slight activity against B. cinerea Pers. The inhibitory indices of them at 1000 mg/L were 26.8% and 33.5%, while the inhibitory index of chitosan was 45.4% [28]. It could be seen that both ␣-ATPMCS and ␣-ATPECS, exhibiting promising antifungal activities even better than that of the fungicide polyoxin. At the concentration of 250 mg/L, ␣-ATPMCS and ␣-ATPECS inhibited growth of P. asparagi, and F. oxysporum at 100%, which are obviously better than that of polyoxin (32.1% against P. asparagi, and 37.2% against F. oxysporum at 250 mg/L). However, polyoxin was more active in controlling S. solani (100% against S. solani) than ␣-ATPMCS (34.5%) and ␣-TPECS (30.6%) at 250 mg/L. For R. solani, it was almost equally sensitive to polyoxin (55.4%), ␣-ATPMCS (47.1%) and ␣-ATPECS (46.5%). It is very interesting to note that ␣-ATPMCS and ␣-ATPECS still showed good activities against P. asparagi, and F. oxysporum at lower concentrations (Fig. 6). F. oxysporum was the most sensitive fungus among the tested fungi to ␣-ATPMCS and ␣-ATPECS. At 31.25 mg/L, the inhibitory index of ␣-ATPMCS and ␣-ATPECS against F. oxysporum was 27.5% and 24.8%, much stronger than that of polyoxin (8.5%). From the above bioassay results, we found that (4-tolyloxy)pyrimidyl-␣-aminophosphonates chitosan derivatives obvious inhibited the selected fungi compared with chitosan. Both ␣ATPMCS and ␣-ATPECS had broad-spectrum antifungal activity. 3.3. Initial mechanism of action studies The cytoplasm and membrane of micro-organisms is the target for many inhibition agents [29–31]. When the membranes become compromised by interaction with antimicrobial agents, low molecular mass species such as K+ and PO4 3− leach out and then followed by DNA, RNA and other materials. Although the actual antimicrobial mechanism of chitosan has not yet fully understood, but it has been suggested to involve with breakdown of the membrane barrier [32–36]. So in this study, to gain insight into the mode of action of the chitosan derivatives, ␣-ATPMCS and ␣-ATPECS were tested for their capacity of affecting membrane permeability of F. oxysporum. Their effect on the relative permeability rate of cell membrane and mycelial pyruvate content was determined. It was shown the images of mycelia obtained from the edge of F. oxysporum culture in the blank control (untreated samples), demonstrating hyphae with typical smooth surface (Fig. 7). While samples treated with ␣-ATPMCS and ␣-ATPECS, the hyphae lost their smoothness and formed abnormal bulges and branches on the surface of fungal hyphae, indicating that inhibited the growth of F. oxysporum by deforming the structure of fungal hyphae. SEM observations of mycelia from the colonies displayed branched conidia with round and chain-like structures. While treated with ␣-ATPMCS and ␣-ATPECS, germination of F. oxysporum conidia was partly inhibited and conidial development of F. oxysporum was suppressed. The results suggest that ␣-ATPMCS and ␣-ATPECS can distort and damage the conidia of F. oxysporum. The effect of ␣-ATPMCS and ␣-ATPECS on membrane permeability was detected in F. oxysporum at 100 mg/L, respectively. When the treatment time was lower than 20 min, relative conductivity with ␣-ATPMCS and ␣-ATPECS treatment was found to be lower
Fig. 9. Change of pyruvic acid of F. oxysporum treated with ␣-ATPMCS and ␣-ATPECS.
than that treated with polyoxin. The results may be attributed to the fact that polyoxin as a small molecule destroyed the cell membrane more quickly than ␣-ATPMCS and ␣-ATPECS. When the treatment time was longer than 20 min, the relative conductivity with ␣ATPECS treatment was higher than that of polyoxin (Fig. 8). It is inferred that this kind of chitosan derivatives may interact with the membrane and cause the leach out of low molecular nucleic acid, proteins, and so on. Fig. 9 showed the change of pyruvic acid of F. oxysporum treated with ␣-ATPMCS and ␣-ATPECS. It was observed that pyruvic acid of F. oxysporum treated with the samples are all lower than the blank control. It may be attributed to their inhibitory effect on the activity of lactate dehydrogenase, thereby inducing the decrease of the pyruvate content [37,38]. Additionally, pyruvic acid of F. oxysporum treated with ␣-ATPMCS and ␣ATPECS was lower than polyxin treated, which was in agreement with the antifungal results. Mycelial pyruvate plays an important role in energy metabolism of carbon source pathway. In this way, it was indicated the chitosan derivatives interfere with the construction of cell architecture and caused hypha growth inhibition. On the basis of the above results, it was concluded that ␣-ATPMCS and ␣-ATPECS executed antifungal properties may associated with energy metabolism and cell wall-degrading. 4. Conclusions In an attempt to discover novel antifungal structures chitosan derivatives with significant activities against crop-threatening fungi, a series of (4-tolyloxy)-pyrimidyl-␣-aminophosphonates chitosan derivatives were prepared. Their structures were confirmed byFT-IR, 13 C NMR, 31 P NMR, elemental analysis, XRD, DSC, and SEM. Antifungal activity of them against S. solani, R. solani, P. asparagi, and F. oxysporum was evaluated. In general, ␣-ATPMCS and ␣-ATPECS obvious inhibited the tested fungi compared with chitosan. And both of them had broad-spectrum antifungal activity. P. asparagi and F. oxysporum were more sensitive to the chitosan derivatives than S. solani and R. solani. According with the preliminary results in these assays, ␣-ATPMCS and ␣-ATPECS even showed much stronger inhibitory effects on P. asparagi and F. oxysporum than the commercial fungicide polyoxin. Additionally, initial mechanism of action studies exhibited ␣-ATPMCS and ␣-ATPECS may affect membrane permeability of F. oxysporum and inhibit mycelial pyruvate content of the fungus. It is important to notice that the derivatives might be good candidates for further design of antifungal agents.
Y. Qin et al. / International Journal of Biological Macromolecules 63 (2014) 83–91
Acknowledgements The study was supported by the Public Science and Technology Research Funds Projects of Ocean (No. 201305016-2), the Science and Technology Development Program of Shandong Province (No. 2012GHY11530), the National High Technology Research and Development Program (“863” Program) of China (2011AA09070405), and the Innovational Foundation of Chinese Academy of Sciences (KZCX2-EW-Q214). References [1] R.A.A. Muzzarelli, J. Boudrant, D. Meyer, N. Manno, M. DeMarchis, M.G. Paoletti, Carbohydr. Polym. 87 (2012) 995–1012. [2] M.N.V.R. Kumar, React. Funct. Polym. 46 (2000) 1–27. [3] T.S. Anirudhan, S. Sandeep, Polym. Chem. UK 2 (2011) 2052–2061. [4] R.S. Jagadish, K.N. Divyashree, P. Viswanath, P. Srinivas, B. Raj, Carbohydr. Polym. 87 (2012) 110–116. [5] S.L. Kosaraju, R. Weerakkody, M.A. Augustin, J. Agric. Food Chem. 58 (2010) 12449–12455. [6] E.I. Rabea, M.E.-T. Badawy, C.V. Stevens, G. Smagghe, W. Steurbaut, Biomacromolecules 4 (2003) 1457–1465. ˜ [7] A.N.H.-L.S. Bautista-Banos, M.G. Velázquez-del Valle, M. Hernández-López, E. Ait Barka, E. Bosquez-Molina, C.L. Wilson, Crop Prot. 25 (2006) 108–118. [8] C.K.S. Pillai, W. Paul, C.P. Sharma, Prog. Polym. Sci. 34 (2009) 641–678. [9] K.V. Harish Prashanth, R.N. Tharanathan, Trend Food Sci. Technol. 18 (2007) 117–131. [10] M.N.V.R. Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A.J. Domb, Chem. Rev. 104 (2004) 6017–6084. [11] V.K. Mourya, N.N. Inamdar, React. Funct. Polym. 68 (2008) 1013–1051. [12] R. Belalia, S. Grelier, M. Benaissa, V. Coma, J. Agric. Food Chem. 56 (2008) 1582–1588. [13] I. Sebti, E. Chollet, P. Degraeve, C. Noel, E. Peyrol, J. Agric. Food Chem. 55 (2007) 693–699.
91
[14] S. De Lombaert, L.B. Stamford, L. Blanchard, J. Tan, D. Hoyer, C.G. Diefenbacher, D. Wei, E.M. Wallace, M.A. Moskal, P. Savage, A.Y. Jeng, Bioorg. Med. Chem. Lett. 7 (1997) 1059–1064. [15] A. Mucha, P. Kafarski, Ł. Berlicki, J. Med. Chem. 54 (2011) 5955–5980. [16] G. Forlani, A. Occhipinti, Ł. Berlicki, G. Dziedzioła, A. Wieczorek, P. Kafarski, J. Agric. Food Chem. 56 (2008) 3193–3199. [17] Z. Zhong, P. Li, R. Xing, X. Chen, S. Liu, Int. J. Biol. Macromol. 45 (2009) 255–259. [18] R.J. Milling, C.J. Richardson, Pestic. Sci. 45 (1995) 43–48. [19] D. Errampalli, N.R. Brubacher, Biol. Control 36 (2006) 49–56. [20] Y. Qin, S. Liu, R. Xing, H. Yu, K. Li, X. Meng, R. Li, P. Li, Carbohydr. Polym. 89 (2012) 388–393. [21] F. Liu, Y. Huang, Pestic. Biochem. Phys. 101 (2011) 248–255. [22] H. Saito, R. Tabeta, K. Ogawa, Macromolecules 20 (1987) 2424–2430. [23] C. Lefay, Y. Guillaneuf, G. Moreira, J.J. Thevarajah, P. Castignolles, F. Ziarelli, E. Bloch, M. Major, L. Charles, M. Gaborieau, D. Bertin, D. Gigmes, Polym. Chem. UK 4 (2013) 322–328. [24] R. Zeng, H. Fu, Y. Zhao, Macromol. Rapid Commun. 27 (2006) 548–552. [25] Y. Wu, Y. Zheng, W. Yang, C. Wang, J. Hu, S. Fu, Carbohydr. Polym. 59 (2005) 165–171. [26] G.O. Phillips, S. Takigami, M. Takigami, Food Hydrocolloids 10 (1996) 11–19. [27] Y. Qin, R. Xing, S. Liu, K. Li, X. Meng, R. Li, J. Cui, B. Li, P. Li, Carbohydr. Polym. 87 (2012) 2664–2670. [28] Z. Guo, R. Xing, S. Liu, Z. Zhong, X. Ji, L. Wang, P. Li, Carbohyd. Res. 342 (2007) 1329–1332. [29] Y. Shai, Pept. Sci. 66 (2002) 236–248. [30] I. Kubo, K.i. Fujita, S.H. Lee, J. Agric. Food Chem. 49 (2001) 1607–1611. [31] B. Hwang, J.-S. Hwang, J. Lee, D.G. Lee, Biochem. Biophys. Res. Commun. 400 (2010) 352–357. [32] X. Li, X. Feng, S. Yang, G. Fu, T. Wang, Z. Su, Carbohydr. Polym. 79 (2010) 493–499. [33] I.M. Helander, E.L. Nurmiaho-Lassila, R. Ahvenainen, J. Rhoades, S. Roller, Int. J. Food Microbiol. 71 (2001) 235–244. [34] M. Kong, X.G. Chen, K. Xing, H.J. Park, Int. J. Food Microbiol. 144 (2010) 51–63. [35] A. El Ghaouth, J. Arul, A. Asselin, N. Benhamou, Mycol. Res. 96 (1992) 769–779. [36] J. Je, S. Kim, J. Agric. Food Chem. 54 (2006) 6629–6633. [37] F. Liu, X. Luo, B. Song, P. Bhadury, S. Yang, L. Jin, W. Xue, D. Hu, Bioorg. Med. Chem. 16 (2008) 3632–3640. [38] W. Xu, S. Yang, P. Bhadury, J. He, M. He, L. Gao, D. Hu, B. Song, Pestic. Biochem. Phys. 101 (2011) 6–15.
