Acyl Chitosan Derivatives - American Chemical Society

Moreover, the European directives for registration of pesti- cides Dir. ...... 1979, 3, 285. (45) Hon, D. N. S. In Polysaccharides in Medicinal Applic...
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Biomacromolecules 2004, 5, 589-595

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Synthesis and Fungicidal Activity of New N,O-Acyl Chitosan Derivatives Mohamed E. I. Badawy,† Entsar I. Rabea,† Tina M. Rogge,‡ Christian V. Stevens,*,‡ Guy Smagghe,† Walter Steurbaut,† and Monica Ho¨fte† Department of Crop Protection, and Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Gent, Belgium Received October 26, 2003; Revised Manuscript Received December 15, 2003

Novel N,O-acyl chitosan (NOAC) derivatives were synthesized to examine their fungicidal activity against the gray mould fungus Botrytis cinerea (Leotiales: Sclerotiniaceae) and the rice leaf blast fungus Pyricularia oryzae (Teleomorph: Magnaporth grisea). The fungicidal activity was evaluated by the radial growth bioassay. NOAC derivatives were more active against the two plant pathogens than chitosan itself, and the effect was concentration dependent. Against B. cinerea, 4-chlorobutyryl chitosan (EC50 ) 0.043%), decanoyl chitosan (EC50 ) 0.044%), cinnamoyl chitosan (EC50 ) 0.045%), and p-methoxybenzoyl chitosan (EC50 ) 0.050%) were the most active (12-13-fold more active than chitosan). (Un)-substituted benzoyl chitosan derivatives were more active against B. cinerea than most of these with N,O-alkyl derivatives. Against P. oryzae chitosan derivatives with lauroyl, methoxy acetyl, methacryloyl and decanoyl were the most active. 1. Introduction Chitosan is a nontoxic, biocompatible, and biodegradable polymer that is prepared by N-deacetylation of chitin. Chitosan consists of β-1,4-linked 2-amino-2-deoxy-D-glucopyranose units and contains no or small amounts of N-acetylD-glucosamine units. Chitosan has become of great interest not only as an underutilized resource but also as a new functional material with high potential in various fields, such as environmental and biomedical engineering.1-4 Chitosan is insoluble in water and most of the organic solvents, although it is soluble in aqueous diluted acids.1,5 The poor solubility of chitosan is probably the major limiting factor for its utilization, i.e., its application in biology, since many enzyme assays are performed at neutral pH. If water-soluble chitosan would be easily accessible, it is expected that the biological and physiological potential of chitosan would increase dramatically. Therefore, special attention was paid to its chemical modification preparing several chitosan derivatives with a higher solubility in water, such as O,N-carboxymethylchitosan,6 N-carboxymethyl-chitosan,7 O-carboxymethylchitosan,8,9 N-carboxyethyl chitosan,10,11 N-sulfate-chitosan,12 O-sulfate chitosan,13 O-butyryl-chitosan,14 N-methylene phosphonic chitosan,15,16 N-benzyl phosphoryl chitosan,17 hydroxypropyl chitosan,18 N-trimethyl chitosan,19 N-succinyl chitosan,20 and O-succinyl chitosan.21 In addition, the successful preparation of N,O-acyl chitosan (NOAC) in MeSO3H as solvent was performed.22,23 A noteworthy point is that both moderate substitution of N,O* To whom correspondence should [email protected]. † Department of Crop Protection. ‡ Department of Organic Chemistry.

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acyl groups and moderate molecular weight are important factors in obtaining highly biologically active chitosan derivatives. Although the selective O acylation of chitosan in MeSO3H (owing to the salt formation of the primary amino group with MeSO3H) was reported,24 the detailed chemical structure and the protecting effect of MeSO3H on the amino group are not clear yet. The preparation of O,O-didecanoyl chitosan was also reported25 through a protected N-phthaloyl chitosan as intermediate.26 This method, however, needs several steps for the protection and deprotection of the N-phthaloyl groups. Therefore, we choose the selective O acylation of chitosan using the MeSO3H system.22-23 Further, there is a continuing search for novel physiological active compounds with a different mode of action in order to overcome resistance. In addition, compounds should possess a high selectivity lowering the danger for humans, fish, beneficial organisms, and the environment in general. Moreover, the European directives for registration of pesticides Dir. 91/414 and Dir. 94/43 and the “Fifth Environmental Action Program” direct toward the use of new and selective pesticides with a low retention. As a consequence, new chitosan derivatives may serve as good alternatives for broad-spectrum and highly persistent pesticides because they are nontoxic to vertebrates and humans, have a biodegradable matrix, and may possess insecticidal and fungicidal properties.17,27 As biologically active material, chitosan has been shown to be fungicidal against Botrytis cinerea as pre- or postharvest treatment. This casual agent of gray mould attacks leaves, stems, flowers, and fruits of various vegetable crops.28 The frequent development of B. cinerea isolates, resistant to common fungicides and the desire to reduce pesticide use, have led to efforts to develop alternatives.17,27,28

10.1021/bm0344295 CCC: $27.50 © 2004 American Chemical Society Published on Web 01/20/2004

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Scheme 1. Synthetic Route of NOAC Derivatives

In this study, NOAC derivatives are investigated for their fungicidal activity against the gray mould B. cinerea and the rice leaf blast disease Pyricularia oryzae.

Badawy et al.

dissolving these derivatives in 1% acetic acid/water, and the pH was adjusted to 5.5-6.0 with 1 M NaOH.29 PDA and OMA media for B. cinerea and P. oryzae, respectively, containing different concentrations of chitosan or synthesized derivatives ranging between 0 and 0.6% (w/v) were seeded in sterile culture plates (9-cm diameter) and infected with 6-mm-diameter agar plugs taken from the margin of a 7-day old culture of B. cinerea or a 10-day old culture of P. oryzae. For each compound, 4 replicates were used for each fungus per concentration tested. The plates were incubated in the dark at 26 ( 2 °C for B. cinerea and/or 30 ( 2 °C for P. oryzae.30 Growth measurements were determined when the hyphae in the control (0 mg/mL) had grown up to the edge of the plate which was after 7 days for B. cinerea and 10 days for P. oryzae. EC50’s and corresponding 95% confidence limits (CL) were estimated by probit analysis.31 In the case of a concentration-response bioassay, EC50’s (representing the concentration of chitosan derivative that provokes 50% inhibition of hyphal growth) and the corresponding 95% confidence limits (CL) were calculated by probit analysis, that is standard in toxicology analyzing biological activity data obtaining a linear regression between the probability (% growth inhibition) and log(concentration).32 3. Results and Discussion

2. Experimental Section 2.1. Materials. Chitosan of low molecular weight (made from coarse ground crab, 85% degree of deacetylation) was purchased from Sigma-Aldrich Co. (Bornem, Belgium) and was used without further purification. Potato dextrose agar (PDA) was purchased from Oxoid Ltd. (Basingstoke, Hampshire, England) and oatmeal agar (OMA) from Becton Dickinson France S. A. (Le Pont de Clair, France). All acid chlorides were purchased from Sigma-Aldrich Belgium and were used as such (purity: 97-99%). The benzoylated cellulose tubings (Sigma-Aldrich) were used after washing with demineralized water. 2.2. General Methods. 2.2.1. NMR Spectroscopy. 1H NMR measurements were performed on a JEOL A-300 NMR spectrometer under a static magnetic field of 300 MHz, at 25 °C or 70 °C. Chemical shift (δ) is given relative to tetramethylsilane (TMS) as internal standard using 0.5 M CD3COOD in D2O as a solvent. 2.2.2. Preparation of N,O-acyl Chitosan DeriVatiVes. Chitosan derivatives were synthesized as shown in Scheme 1. In brief, 10 mmol of chitosan (1.7 g calculated as glucosamine unit) was dissolved in MeSO3H (20 mL) at room temperature for 1 h. To this solution was added the acyl chloride (1 equiv/glucosamine unit of chitosan). The mixture was stirred at room temperature for 5 h, and 30 g of crushed ice was added to stop the reaction. The acidic mixture was dialyzed for 1 day to remove most of the acid, followed by neutralization with NaHCO3. Finally, the mixture was dialyzed again for more than 3 days and lyophilized.22 2.3. Fungicidal Assessment in a Radial Hyphal Growth Bioassay. Chitosan and NOAC solutions were prepared by

3.1. Synthesis of N,O-Acyl Chitosan Derivatives. Table 1 shows the structures of the new chitosan derivatives using 1 equiv of acyl chloride per repeating unit of original chitosan (NH2 ) 0.85, NHAc ) 0.15). The chitosan derivatives were obtained in moderate to good yields (at least 53%). The molecular fraction (MF) of each functional group was estimated by 1H NMR spectra. The results indicated that the reaction mainly occurred on the OH group and not on the NH2 group. This is shown in Table 1 as MF of NHCOR, which approximately equals zero. These data show that the protection of the amino group by the salt formation with MeSO3H was nearly quantitative (90.6-100%). The MF of OCOR was very diverse and the measurement by 1H NMR was not very accurate ranging from 1.64 to 0.21. 3.2. 1H NMR Measurements. Spectral Data for Compound 1. 1H NMR (25 °C): δ 2.0 (m, 0.45H, NHAc), 3.19 (br s, 0.85H, H-2 of GlcN residue), 3.37 (s, 0.33H, OCH3), 3.62-4.0 (br m, 5.15H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.05 (s, 0.22H, CH2), 4.88 (br s, H-1 of GlcN residue). MF (NH2, x) was estimated from δ 3.19 (x) vs 3.62-4.0 (6 - x). MF (NHAc, y) was estimated from δ 2.0 (3y) vs 3.19-4.0 (6H). MF [(NHCOR + OCOR), z] was estimated from δ 3.37 (3z) vs 3.19-4.0 (6H). MF (NHCOR) ) 1 - x - y; MF (OCOR) ) z - MF (NHCOR). Spectral Data for Compound 2. 1H NMR (25 °C): δ 2.0 (s, 0.30H, NHAc), 2.09 (m, 0.51H, CH3), 3.23 (br s, 0.83H, H-2 of GlcN residue), 3.57-4.15 (m, 5.17H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.92 (br s, H-1 of GlcN residue), 5.83 (br s, 0.17H, CH)), 6.23 (br s, 0.17H, CH)). MF (NH2, x) was estimated from δ 3.23 (x) vs 3.57-4.15 (6 - x). MF (NHAc, y) was estimated from δ 2.0 (3y) vs 3.23-4.15 (6H). MF [(NHCOR + OCOR), z]

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Table 1. Chemical Structure of NOAC Derivatives

molecular fraction (MF)c compound

F.Wa

yield (%)b

NH2 (x)

NHAc (y)

NHCOR

OCOR (z)

OH

protection of NH2 (%)d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

175 177 286 261 195 273 289 210 228 289 219 222 390 256 204 203 205 226

87 80 79 76 62 83 80 81 89 92 78 81 69 74 67 53 73 75

0.85 0.83 0.80 0.77 0.84 0.84 0.80 0.84 0.83 0.83 0.82 0.79 0.78 0.85 0.84 0.84 0.82 0.83

0.15 0.10 0.18 0.17 0.09 0.14 0.19 0.16 0.15 0.13 0.11 0.18 0.17 0.13 0.13 0.16 0.09 0.11

0 0.07 0.01 0.06 0.07 0.02 0.01 0 0.02 0.04 0.07 0.03 0.05 0.02 0.03 0 0.09 0.06

0.11 0.10 1.64 0.82 0.18 1.05 0.70 0.23 0.21 0.42 0.44 0.42 1.59 0.62 0.22 0.18 0.22 0.39

1.89 1.90 0.36 1.18 1.82 0.95 1.30 1.77 1.79 1.58 1.56 1.58 0.41 1.38 1.78 1.82 1.78 1.61

100 97.6 94 90.6 98.8 98.8 94 98.8 97.6 97.6 96.5 93 91.8 100 98.8 98.8 96.5 97.6

a FW ) 161 + [MF of (NHAc) × 43 + MF of (NHCOR + OCOR) × MW of substituent]. b Yield was determined by weight recovery in accordance with the change in FW according to the substitution level (MF) determined by 1H NMR. c MF (NH2, X) was calculated from this equation: B/C ) X/(6 - X), MF of (NHAc, DA): A/(B + C) ) 3DA/6, MF of (NHCOR + OCOR, DS) ) DS: D/(B + C) ) nDS/6, where A is the peak area of NHAc, B is the peak area of H-2 of GlcN unit, C is the peak area of H-2 of GlcNAc and H-3,4,5,6 of GlcN, D is the peak area of substituent, and n is the number of hydrogen atom per substituent. d Protection of NH2 (%) ) [MF of (NH2)/0.85] × 100.

was estimated from δ 2.09 (3z) vs 3.23-4.15 (6H). MF (NHCOR) ) 1 - x - y; MF (OCOR) ) z - MF (NHCOR). Spectral Data for Compound 3. 1H NMR (70 °C): δ 0.93 (br s, 4.95H, CH3), 1.62 [br s, 3.3H, CH2 (β)], 2.22 (br s, 0.54H, NHAc), 2.39 [br s, 3.3H, CH2 (R)], 3.27 (br s, 0.80H, H-2 of GlcN residue), 3.60-4.10 (br m, 5.20H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.97 (br s, H-1 of GlcN residue). MF (NH2, x) was estimated from δ 3.27 (x) vs 3.60-4.10 (6 - x). MF (NHAc, y) was estimated from δ 2.22 (3y) vs 3.27-4.10 (6H). MF [(NHCOR + OCOR), z] was estimated from δ 0.93 (Me, 3z) vs 3.274.10 (6H). MF (NHCOR) ) 1 - x - y; MF (OCOR) ) z MF (NHCOR). Spectral Data for Compound 4. 1H NMR (25 °C): δ 1.72 [q, 1.76H, CH2 (β)], 2.0 (s, 0.51H, NHAc), 2.32 [t, 1.76H, CH2 (R)], 3.16 (br s, 0.77H, H-2 of GlcN residue), 3.52 [t, 1.76H, CH2 (γ)], 3.61-4.0 (br m, 5.23H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.86 (br s, H-1 of GlcN residue). MF (NH2, x) was estimated from δ 3.16 (x) vs 3.61-4.0 (6 - x). MF (NHAc, y) was estimated from δ 2.0

(3y) vs 3.16-4.0 (6H). MF [(NHCOR + OCOR), z] was estimated from δ 1.72 (2z) vs 3.16-4.0 (6H). MF (NHCOR) ) 1 - x - y; MF (OCOR) ) z - MF (NHCOR). Spectral Data for Compound 5. 1H NMR (25 °C): δ 1.30 [s, 1.5H, (CH3)2], 2.0 (s, 0.27H, NHAc), 3.19 (br s, 0.84H, H-2 of GlcN residue), 3.60-4.10 (br m, 5.16H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit and CH2 of the substituent), 4.90 (br s, H-1 of GlcN residue). MF (NH2, x) was estimated from δ 3.19 (x) vs 3.60-4.10 (6 - x). MF (NHAc, y) was estimated from δ 2.0 (3y) vs 3.19-4.10 (6H). MF [(NHCOR + OCOR), z] was estimated from δ 1.30 (Me, 6z) vs 3.19-4.10 (6H). MF (NHCOR) ) 1 - x - y; MF (OCOR) ) z - MF (NHCOR). Spectral Data for Compound 6. 1H NMR (25 °C): δ 0.90 (br s, 3.21H, CH3), 1.30 [br s, 4.28H, (CH2)2], 1.61 [br s, 2.14H, CH2 (β)], 2.04 (s, 0.42H, NHAc), 2.41 [br s, 2.14H, CH2 (R)], 3.20 (br s, 0.84H, H-2 of GlcN residue), 3.654.20 (br m, 5.16H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.90 (br s, H-1 of GlcN residue). MF was estimated in a manner similar to that used for compound 3.

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Spectral Data for Compound 7. 1H NMR (70 °C): δ 0.90 (br s, 2.13H, CH3), 1.27 [br s, 8.52H, (CH2)6], 1.57 [br s, 1.42H, CH2 (β)], 2.03 (s, 0.57H, NHAc), 2.30 [br s, 1.42H, CH2 (R)], 3.17 (br s, 0.80H, H-2 of GlcN residue), 3.604.21 (br m, 5.20H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.90 (br s, H-1 of GlcN residue). MF was estimated in a manner similar to that used for compound 3. Spectral Data for Compound 8. 1H NMR (70 °C): δ 0.89 (br s, 0.69H, CH3), 1.28 [br s, 3.68H, (CH2)8], 1.66 [br s, 0.46H, CH2 (β)], 2.06 (s, 0.48H, NHAc), 2.30 [br s, 0.46H, CH2 (R)], 3.19 (br s, 0.84H, H-2 of GlcN residue), 3.604.10 (br m, 5.16H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.90 (br s, H-1 of GlcN residue). MF was estimated in a manner similar to that used for compound 3. Spectral Data for Compound 9. 1H NMR (25 °C): δ 0.90 (br s, 0.69H, CH3), 1.30 [br s, 5.52H, (CH2)12], 1.65 [br s, 0.46H, CH2 (β)], 2.04 (s, 0.45H, NHAc), 2.30 [br s, 0.46H, CH2 (R)], 3.20 (br s, 0.83H, H-2 of GlcN residue), 3.754.10 (br m, 5.17H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.89 (br s, H-1 of GlcN residue), 5.36 [s, 0.46H, (CHdCH)]. MF was estimated in a manner similar to that used for compound 3. Spectral Data for Compound 10. 1H NMR (70 °C): δ 0.88 (br s, 1.38H, CH3), 1.21 [br s, 13.80H, (CH2)15], 1.40 [br s, 0.92H, CH2 (β)], 2.0 (s, 0.39H, NHAc), 2.50 [br s, 0.92H, CH2 (R)], 3.20 (br s, 0.83H, H-2 of GlcN residue), 3.604.15 (br m, 5.17H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.90 (br s, H-1 of GlcN residue). MF was estimated in a manner similar to that used for compound 3. Spectral Data for Compound 11. 1H NMR (70 °C): δ 2.06 (s, 0.33H, NHAc), 3.20 (br s, 0.82H, H-2 of GlcN residue), 3.50-4.20 (br m, 5.18H, H-2 of N-acylated GlcN and H-3,4,5,6 of of GlcN unit), 4.90 (br s, H-1 of GlcN residue), 7.30-8.20 (m, 2.55H, Ph). MF (NH2, x) was estimated from δ 3.20 (x) vs 3.50-4.20 (6 - x). MF (NHAc, y) was estimated from δ 2.06 (3y) vs 3.20-4.20 (6H). MF [(NHCOR+OCOR), z] was estimated from δ 7.30-8.20 (Ph, 5z) vs δ 3.20-4.20 (6H). MF (NHCOR) ) 1 - x - y; MF (OCOR) ) z - MF (NHCOR). Spectral Data for Compound 12. 1H NMR (70 °C): δ 2.04 (s, 0.54H, NHAc), 2.43 (br s, 1.35H, CH3), 3.20 (br s, 0.79H, H-2 of GlcN residue), 3.60-4.21 (br m, 5.21H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.89 (br s, H-1 of GlcN residue), 7.0-8.0 (br m, 1.80H, Ph). MF was estimated in a manner similar to that used for compound 11. Spectral Data for Compound 13. 1H NMR (25 °C): δ 2.03 (s, 0.51H, NHAc), 3.15 (br s, 0.78H, H-2 of GlcN residue), 3.30-4.20 (br m, 5.22H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit and the OCH3 of the substituent), 4.87 (br s, H-1 of GlcN residue), 6.70-7.10 (br m, 3.28H, Ph), 7.40-8.10 (br m, 3.28H, Ph). MF was estimated in a manner similar to that used for compound 11. Spectral Data for Compound 14. 1H NMR (25 °C): δ 2.07 (s, 0.39H, NHAc), 3.25 (br s, 0.85H, H-2 of GlcN residue), 3.60-4.20 (br m, 5.15H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.92 (br s, H-1 of GlcN residue), 7.10-8.10 (br m, 2.56H, Ph). MF was estimated in a manner similar to that used for compound 11.

Badawy et al.

Spectral Data for Compound 15. 1H NMR (25 °C): δ 2.03 (s, 0.39H, NHAc), 3.23 (br s, 0.84H, H-2 of GlcN residue), 3.70-4.20 (br m, 5.16H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.95 (br s, H-1 of GlcN residue), 8.06 (d, 0.50H, Ph), 8.30 (d, 0.50H, Ph). MF was estimated in a manner similar to that used for compound 11. Spectral Data for Compound 16. 1H NMR (25 °C): δ 2.21 (s, 0.48H, NHAc), 3.19 (br s, 0.84H, H-2 of GlcN residue), 3.60-4.10 (br m, 5.16H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.90 (br s, H-1 of GlcN residue), 9.02 (s, 0.36H, Ph), 9.11 (s, 0.18H, Ph). MF was estimated in a manner similar to that used for compound 11. Spectral Data for Compound 17. 1H NMR (25 °C): δ 2.04 (s, 0.27H, NHAc), 3.23 (br s, 0.82H, H-2 of GlcN residue), 3.50-4.20 (br m, 5.18H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.82 (br s, H-1 of GlcN residue), 6.72 (d, 0.31H, CH), 7.20-7.80 (br m, 1.55H, Ph), 7.90 (d, 0.31H, CH). MF was estimated in a manner similar to that used for compound 11. Spectral Data for Compound 18. 1H NMR (25 °C): δ 2.06 (s, 0.33H, NHAc), 3.22 (br s, 0.83H, H-2 of GlcN residue), 3.70-4.20 (br m, 5.17H, H-2 of N-acylated GlcN and H-3,4,5,6 of GlcN unit), 4.55 (s, 0.90H, CH2), 4.93 (br s, H-1 of GlcN residue), 6.95-7.10 (m, 1.35H, Ph), 7.42 (t, 0.90H, Ph). MF was estimated in a manner similar to that used for compound 11. 3.3. Fungicidal Activity of Synthesized Compounds against B. cinerea. The fungicidal activity of NOAC derivatives against the gray mould B. cinerea as EC50 values with 95% confidence limits was achieved by the radial growth bioassay (Table 2). One can see that the antifungal activity of the synthesized derivatives is higher than that of chitosan itself, except for stearoyl chitosan (10). It was clear that the most active compounds in all alkyl derivatives (110) were 4-chlorobutyryl chitosan (4) and decanoyl chitosan (7), with an EC50 of 0.043% (Figure 1A) and 0.044%, respectively. The activity of both compounds was approximately 13 times higher than chitosan. For compound 10, the activity was low (EC50 0.602%); however, we believe this may be due to a poor solubility. In summary, our results demonstrated that the toxicity was decreasing when the alkyl chain was higher than C10 as shown in compounds 8-10; this conclusion confirms the previous data that the alkyl chain length strongly affects the antimicrobial activity of the chitosan derivatives, where N-propyl-N,N-dimethyl chitosan was stronger than N,N,Ntrimethyl chitosan against Escherichia coli.33-34 According to the literature,35 a halogen substitution on the phenyl ring may play an important role in the biological activity. Herein, we noticed that the presence of the chlorine atom on the alkyl group (4 and 5) led to a significant increase of the activity against B. cinerea. This was very clear for compound 4 with an EC50 of 0.043% vs butyryl chitosan (3) with an EC50 of 0.27% and also for 3-chloropivaloyl chitosan (5) compared to compound 3. This conclusion was also found in a previous experiment where N-2,6-dichlorobenzyl chitosan was the most active against B. cinerea as compared to other substituted benzyl chitosan derivatives.27 On the other hand, when a phenyl ring replaced the alkyl

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Table 2. Fungicidal Activity of Chitosan and NOAC Derivatives against B. Cinerea in a Standardized Radial Hyphal Growth Bioassay compound

EC50 (95 CL) (%, v/v)

probit regressiona

chitosan 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0.562 (0.409-0.771) 0.105 (0.094-0.118) 0.051 (0.042-0.061) 0.272 (0.213-0.347) 0.043 (0.033-0.056) 0.070 (0.057-0.085) 0.088 (0.069-0.113) 0.044 (0.029-0.065) 0.113 (0.098-0.131) 0.117 (0.107-0.129) 0.602 (0.452-0.801) 0.059 (0.046-0.075) 0.070 (0.058-0.085) 0.050 (0.041-0.061) 0.104 (0.092-0.117) 0.094 (0.082-0.109) 0.078 (0.064-0.094) 0.045 (0.037-0.055) 0.096 (0.081-0.114)

Y ) -4.21 + 1.12X; χ2 ) 0.99, P ) 0.96, df ) 5 Y ) -7.91 + 2.62X; χ2 ) 6.61, P ) 0.16, df ) 4 Y ) -7.52 + 2.77X; χ2 ) 1.01, P ) 0.60, df ) 2 Y ) -4.30 + 1.25X; χ2 ) 2.04, P ) 0.45, df ) 3 Y ) -5.92 + 2.24X; χ2 ) 3.04, P ) 0.22, df ) 2 Y ) -5.56 + 1.95X; χ2 ) 7.23, P ) 0.13, df ) 3 Y ) -3.96 + 1.34X; χ2 ) 0.75, P ) 0.68, df ) 2 Y ) -3.42 + 1.29X; χ2 ) 16.21, P ) 0.09, df ) 3 Y ) -6.49 + 2.13X; χ2 ) 1.96, P ) 0.37, df ) 2 Y ) -10.13 + 3.30X; χ2 ) 2.11, P ) 0.35, df ) 2 Y ) -12.69 + 3.35X; χ2 ) 0.81, P ) 0.66, df ) 2 Y ) -4.92 + 1.77X; χ2 ) 0.23, P ) 0.89, df ) 2 Y ) -5.91 + 2.07X; χ2 ) 13.26, P ) 0.07, df ) 3 Y ) -4.55 + 1.68X; χ2 ) 8.2, P ) 0.06, df ) 3 Y ) -8.40 + 2.78X; χ2 ) 5.0, P ) 0.08, df ) 2 Y ) -6.78 + 2.28X; χ2 ) 7.95, P ) 0.07, df ) 3 Y ) -5.59 + 1.93X; χ2 ) 2.92, P ) 0.23, df ) 2 Y ) -4.63 + 1.74X; χ2 ) 5.19, P ) 0.19, df ) 3 Y ) -5.64 + 1.89X; χ2 ) 1.84, P ) 0.61, df ) 3

a The probit regression line Y ) a + bX with Y ) probit(% hyphal growth inhibition) and X ) logarithm(concentration in the medium) (%, w/v) and corresponding χ2, p value and df, was calculated in accord with Finney (1971).

Figure 1. Effects of chitosan derivatives on the hyphal growth of B. cinerea. (A) p-Chlorobutyryl chitosan (4), (B) cinnamoyl chitosan (17). In A and B, from left to right 0.4, 0.2, 0.1, and 0.05% (w/v) and the control. In C, effect of p-chlorobenzoyl chitosan (14) at 0.05% (left) on spore formation compare to control (right). f: Arrows indicate spore formation that was not seen in the treatment.

chain, p-chlorobenzoyl chitosan (14) had the lowest activity compared to the other phenyl substituted chitosan derivatives. With the aromatic substituents, cinnamoyl chitosan (17) (Figure 1B) and p-methoxybenzoyl chitosan (13) were the most active compounds against B. cinerea (about 12-fold higher than chitosan). 3,5-Dinitrobenzoyl chitosan (16) was more potent than the one with the p-nitro substituent (15). The data also showed that p-methoxy benzoyl chitosan was more active (about 1.5-fold) than p-methylbenzoyl chitosan. In addition, as shown in Figure 1C, most of these derivatives prevent spore formation compared to the control.

In general, chitosan inhibits spore germination and radial growth of B. cinerea and Rhizopus stolonifer.30 However, this author did not achieve complete inhibition even at a concentration of 0.6%, indicating that chitosan is more fungistatic rather than fungicidal. In addition, the effectiveness of pre- and post-harvest treatments with chitosan (0.1, 0.5, and 1.0%) to control B. cinerea on grapes was investigated.36 In post-harvest treatments, small bunches dipped in chitosan solutions and inoculated with the pathogen showed a reduction of incidence, severity, and nesting of gray mould, in comparison with the control. Single berries

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Figure 2. Fungicidal activity of chitosan and NOAC derivatives (1-18) against P. oryzae at 0.5%, w/v. Chitosan derivatives were prepared in OMA culture medium and then tested in a radial growth bioassay. Data are expressed as mean percentages ( SEM based on 4 replicates per tested concentration.

artificially wounded, treated with the polymer, and inoculated with B. cinerea showed a reduced percentage of infected berries and lesion diameters. All pre-harvest treatments significantly reduced the incidence of gray mould, as compared to the control. Grapes treated with 1% chitosan showed a significant increase of phenylalanine ammonia-lyase activity. Consequently, besides a direct activity against B. cinerea, chitosan produces other effects contributing to reduce decay.36 Five chemically modified chitosans were tested for their antifungal activities against Saprolegnia parasitica in a fungal growth assay with agar and broth media.37 Results indicated an abnormal growth as was observed on the first day, for methyl pyrolidinone chitosan and N-phosphonomethyl chitosan, and on the second day, for N-carboxymethyl chitosan, with a tightly packed precipitate present on the bottom of the test tubes. In contrast, controls showed a fluffy fungal material. Further on, N-dicarboxymethyl chitosan favored fungal growth, and dimethylaminopropyl chitosan had no significant effect over the controls.37 Based on our experiments and those from literature, we believe that the use of natural compounds to control plant pathogens may lead to a reduction in the use of fungicides. Chitosan is already known to interfere with the growth of several phytopathogenic fungi including B. cinerea, but the mechanism by which it affects the growth of the pathogen is still unclear.38,39 It is believed that chitosan interferes with the negatively charged residues of macromolecules exposed on the fungal cell surface and thereby changing the permeability of the plasma membrane.4,40 3.4. Fungicidal Activity of Synthesized Compounds against P. oryzae. P. oryzae, the causal agent of rice blast disease, is a filamentous heterothallic Ascomycete with septate hyphae that contains a single nucleus per cell. When P. oryzae infects rice plants, rice blast disease appears as brown spots in ears, leaves, and stems of rice plants and leads to severe damage to the rice plants, resulting in a distinctive reduction of the grain yield.41-42 Figure 2 shows the fungicidal activity of NOAC derivatives against P. oryzae at 0.5%. The results indicated that all of the synthesized compounds were more potent than the original chitosan at the same concentration. Chitosan in this

case showed no activity against this fungus. This result is in agreement with Liu et al.,43 reporting a minimum growth inhibitory concentration (MIC) of 0.5% of chitosan against P. oryzae. It was very clear that the most active one in these derivatives was lauroyl chitosan (8), 70.26% inhibition of hyphal growth at 0.5%, which means about a 14-fold higher activity than chitosan. In addition, with a range of lower concentrations (0.05, 0.1, 0.2, and 0.4%) of this compound, 17, 20, 38, and 55% inhibition, respectively, was achieved. Using Finney (1971), the estimated EC50 (95% CL) of compound 8 was 0.278 (0.227-0.341) % (w/w), and the probit (%mortality) - log(concentration) line, Y ) -4.89 + 1.42X, χ2 ) 3.5, P ) 0.16, df, 3. Methacryloyl chitosan (2), decanoyl chitosan (7), and methoxyacetyl chitosan (1) are following compound 8 in the descending order of the activity (66.67, 64.71, and 64.29% inhibition of hyphal growth of the tested fungus, respectively). The result also indicated that in the alkyl substituents (1-10) the moderate active compounds were oleoyl chitosan (9) and stearoyl chitosan (10). However, the less toxic one in this group was hexanoyl chitosan (6). The toxicity dramatically decreased by replacing the lauroyl chain (8) by a phenyl ring (11). From our results, we noticed that most of these derivatives with an alkyl chain (1-10) were more active than these with an aromatic substituent (11-18). P-Chlorobenzoyl chitosan (14) was the most active compound in the aromatic series. In addition, the moderate activity in this group was found with phenoxyacetyl chitosan (18). However, p-nitrobenzoyl chitosan (15) was the least potent one in all NOAC derivatives against the tested fungus. Based on our experiments, chitosan and its NOAC derivatives showed low biological activity against P. oryzae (Ascomycetes). This can be explained by the fact that this fungus contains chitin and chitosan as major components of its cell wall and septa as previously discussed in the literature.44-46 4. Conclusion From this study, we can conclude that the reaction of acyl chlorides with chitosan resulted in O-acetylated chitosan as a major product compared to the N-acetylated chitosan, after

New N,O-Acyl Chitosan Derivatives

protection of NH2 with MeSO3H. In general, the acyl chitosan derivatives have a highly improved antifungal activity against the gray mold B. cinerea and the blast mould P. oryzae compared to the original chitosan. Results on antifungal activity of chitosan and N,O-acyl chitosans against the two pathogenic fungi showed that all derivatives were more active against B. cinerea than against P. oryzae. Chitosan itself had negligible activity against B. cinerea (EC50 0.56%) and showed no toxicity against the second fungus at higher concentration (0.5%). Acknowledgment. This research was supported by a doctoral fellowship from the Egyptian government to E.I. Rabea. References and Notes (1) Dodane, V.; Vilivalam, V. D. Pharm. Sci. Technol. Today 1998, 1, 246. (2) Loke, W. K.; Lau, S. K.; Yong, L. L.; Khor, E.; Sum, C. K. J. Biomed. Mater. Res. (Appl. Biomater.) 2000, 53, 8. (3) Denkbas, E. B.; Odabasi, M. J. Appl. Polym. Sci. 2000, 76, 1637. (4) Rabea, E. I.; Badawy, M. E.-T.; Stevens, C. V.; Smagghe, G.; Steurbaut, W. Biomacromolecules 2003, 4, 1457. (5) Hirano S, Yamaguchi Y.; Kamiya M. Carbohydr. Polym. 2002, 48, 203. (6) Marguerite, R.; Pham, L. D.; Claude, G. Int. J. Biol. Macromol. 1992, 14, 122. (7) Muzzarelli, R. A. A.; Tanfani, F. Carbohydr. Polym. 1985, 5, 297. (8) Riccardo, A.; Muzzarelli, A. Carbohydr. Polym. 1988, 8, 1. (9) Lillo, L. E.; Matsuhiro, B. Carbohydr. Polym. 1997, 34, 397. (10) Sashiwa, H.; Kawasaki, N.; Nakayama, A.; Muraki, E.; Yajima, H. Yamamori, N.; Ichinose, Y. Sunamoto, J.; Aiba, S. Carbohydr. Res. 2003, 338, 557. (11) Skorik, Y.; Gomes, C. A. R.; Vasconcelos, T. S. D.; Yatluk, Y. G. Carbohydr. Res. 2003, 338, 271. (12) Holme, K. R.; Perlin, A. S. Carbohydr. Res. 1997, 302, 173. (13) Focher, B.; Massoli, A.; Torri, G.; Gervasini, A.; Morazzoni, F. Macromol. Chem. 1986, 187, 2609. (14) Grant, S.; Blair, H.; Mckay, G. Polym. Commun. 1988, 29, 342. (15) Heras, A.; Rodriguez, N. M.; Ramos, V. M.; Agullo, E. Carbohydr. Polym. 2001, 44, 1. (16) Ramos, V. M.; Rodriguez, N. M.; Diaz, M. F.; Rodriguez, M. S.; Heras, A.; Agullo, E. Carbohydr. Polym. 2003, 52, 39. (17) Rabea, E. I.; Badawy, M. E.-T.; Rogge, T. M.; Stevens, C. V.; Smagghe, G.; Ho¨fte, M.; Steurbaut, W. In Proceedings of the 9th International Chitin-Chitosan Conference; Montereal, Que´bec, Canada 2003; p 103. (18) Wenming, X.; Peixin, X.; Qin, L. Carbohydr. Polym. 2002, 50, 35. (19) Zhishen, J.; Dongfen, S.; Weiliang, X. Carbohydr. Res. 2001, 333, 1.

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