Synthesis and Antimicrobial Activity of N-(6-Carboxyl Cyclohex-3-ene Carbonyl) Chitosan with Different Degrees of Substitution

Five products of N-(6-carboxyl cyclohex-3-ene carbonyl) chitosan as antimicrobial agents were prepared by reaction of chitosan with tetrahydrophthalic anhydride (THPA) at different degrees of substitution (DS).The antimicrobial activitywas evaluated against four plant bacteria and eight fungi. The results proved that the inhibitory property and water solubility of the synthesized chitosan derivatives, with increase of the DS, exhibited a remarkable improvement over chitosan.The product with a DS of 0.40 was themost active one with MIC of 510, 735, 240, and 385mg/L against Erwinia carotovora, Ralstonia solanacearum, Rhodococcus fascians, and Rhizobium radiobacter, respectively, and also in mycelial growth inhibition againstAlternaria alternata (EC 50 = 683mg/L), Botrytis cinerea (EC 50 = 774mg/L), Botryodiplodia theobromae (EC 50 = 501mg/L), Fusarium oxysporum (EC 50 = 500mg/L), F. solani (EC 50 = 260mg/L), Penicillium digitatum (EC 50 = 417mg/L), Phytophthora infestans (EC 50 = 298mg/L), and Sclerotinia sclerotiorum (EC 50 = 763mg/L). These compounds based on a biodegradable and biocompatible chitosan could be used as potentially antimicrobial agents in crop protection instead of hazardous synthetic pesticides.

In agriculture, pre-and postharvest protection from different pathogens rely mainly on synthetic fertilizers and hazardous pesticides.Therefore, alternative solutions are needed due to concerns for public health, environmental protection, and development of resistant pests.Chitosan is widely used as a biocide either alone or blended with other natural products against plant pathogens that induce decay and harmful effects of the agricultural crops during the growing season and postharvest phase [5,6,[11][12][13].The antimicrobial activity of chitosan is coming from its polycationic nature and with chemical modification [5,11,12,14,15].However, its low solubility at the pH higher than of 6.5 is the major limiting factor in its utilization, that is, its application in biology, since many enzyme assays are performed in neutral media.Therefore, the preparation of water-soluble chitosans could enhance its biological and physiological potential [5,11,15,16].From the synthetic point, all of the chemical modifications have been performed on the primary amino group (at C-2) or the two hydroxyl groups (at C-3 and C-6) of the chitosan molecule such as N-reductive amination, quaternization, hydroxyalkylation, N,O-carboxymethylation, 2 International Journal of Carbohydrate Chemistry N,O-acylation, phosphorylation, N,O-phthaloylation, N,Osuccinylation, chitosan-amino acid and chitosan-peptide conjugates, and graft copolymerization [17][18][19][20][21][22][23].Acylation of chitosan was the typical method involves reacting chitosan with either an acid chloride or acid anhydride forming N-(acyl) chitosan, O-(acyl) chitosan, N,O-(acyl), N-(hydroxyacyl) chitosan, or N-(carboxyacyl) chitosan derivatives, but the reactions are not regioselective partly because of the heterogeneous reaction conditions [24][25][26].
Based on the current state of research and progress in corresponding areas, this article aims at preparing water-soluble N-(6-carboxyl cyclohex-3-ene carbonyl) chitosan derivatives to enhance the antimicrobial activity of chitosan molecule against plant pathogenic bacteria Erwinia carotovora, Ralstonia solanacearum, Rhodococcus fascians, and Rhizobium radiobacter.In addition, the antifungal activity was tested against Alternaria alternata, Botrytis cinerea, Botryodiplodia theobromae, Fusarium oxysporum, Fusarium solani, Penicillium digitatum, Phytophthora infestans, and Sclerotinia sclerotiorum.The antimicrobial activities as MICs for bacteria and EC 50 s for fungi were investigated and discussed in detail.

Average Molecular Weight (MW).
The average MWs of chitosan products were obtained from Mark-Houwink equation ( =   ) [34,35], where  is the intrinsic viscosity and  and  are viscometric parameters depending on the solvent.For a chitosan dissolved in 0.5 M CH 3 COOH/0.2M CH 3 COONa buffer,  and  are found to be 3.5 × 10 −4 and 0.76, respectively [36,37].The viscosity of chitosan solution of different concentrations in acetate buffer (pH = 5) was measured by using Ubbelohde Viscometer (capillary section size 0.7 mm) immersed in a constant temperature bath at 25 ∘ C and having flow time for buffer solution,  0 = 53.65 seconds.During preparation, all the solutions were magnetically stirred for 1 h to ensure proper dissolution of chitosan and were filtered using Whatman filter paper number 4. The flow times of chitosan solutions and solvent were recorded in triplicate and the average value was calculated.The intrinsic viscosity [] was calculated graphically by extrapolating the curve of reduced viscosity versus chitosan concentration (g/dL) to zero concentration.The MW was then calculated by using Mark-Houwink equation.

Solubility Test.
The solubility of chitosan and N-(6carboxyl cyclohex-3-ene carbonyl) chitosan products was performed in distilled water, aqueous acetic acid (0.1, 0.5, and 1%, v/v), and 1% NaOH (w/v).A sample was soaked in each solvent at the concentration of 10 mg/mL and the solubility was checked after standing for 24 h at room temperature [38].

Antifungal Activity.
The activity was tested using mycelia radial growth technique [41].The compounds were dissolved as described in the antibacterial assay and serial concentrations of 250, 500, 1000, 1500, 2000, 2500, and 3000 mg/L were tested.The aliquots of the stock solutions were added to the PDA medium and then transferred to Petri dishes.After solidification, the mixtures were inoculated with a 5 mm in diameter mycelium fungi at the center of Petri dishes and these were incubated in the dark at 27 ± 2 ∘ C. The fungal growth was measured when the control had grown to the edge of the plate.The inhibition of fungal growth was calculated as the percentage of inhibition of radial growth compared to the control.The effective concentration that inhibits 50% of mycelial growth (EC 50 ) for each compound was estimated by probit analysis [42] using of SPSS 21.0 software.a THPA = tetrahydrophthalic anhydride.b GlcN = glucosamine.c DA is a degree of acetylation (calculated as 1 − DDA).d DDA is a degree of deacetylation.
e DS is a degree of substitution.f FW is a formula weight.g MW is the molecular weight in g/mol and it was determined for chitosan by measuring its intrinsic viscosity according to the Mark-Houwink-Sakurada equation and it was calculated for compounds 1-5 as follows: 2.9.Statistical Analysis.Statistical analysis was performed using SPSS 21.0 software (Statistical Package for Social Sciences, USA).The log dose-response curves allowed determination of the EC 50 values for the fungal bioassay according to the probit analysis [42].The 95% confidence limits for the range of EC 50 values were determined by the least-square regression analysis of the relative growth rate (% control) against the logarithm of the compound concentration.

Characterization of N-(6-Carboxyl Cyclohex-3-ene Carbonyl)
Chitosans.The chemical structure, degree of deacetylation (DDA), degree of substitution (DS), and formula weights (FW) were estimated by 1 H-NMR spectra [17,43] and the data are presented in Table 1.Chitosan products were obtained at a range of DS 0.09-0.40when five mole ratios (0.1, 0.3, 0.5, 0.8, and 1.0 mole) of THPA per glucose amine were tested.A hydrogel was produced in the reaction mixture with each product.The products of N-(6-carboxyl cyclohex-3-ene carbonyl) chitosans were obtained with 84.79, 86.01, 94.86, 96.43, and 97.11% yield at DS of 0.09, 0.18, 0.26, 0.30, and 0.40, respectively.Figure 2 shows the 1 H-and 13 C-NMR spectra of chitosan and compound 5.The peak at  2.07-2.12ppm is assigned to the proton of residual CH 3 in acetyl group (Figures 2(a) and 2(b)).The peak at  3.09-3.30ppm is attributed to H-2 of GlcN residue.The broad multiple peak at  3.49-4.15ppm is assigned for H-3,4,5,6 of GlcN unit and H-2,3,4,5,6 of GlcNAc unit.The intense band at 4.8-5.30ppm is related to OH groups and HDO (solvent).In this region, as observed more clearly from an extended spectrum, some different anomeric protons (H-1 of GlcN and GlcNAc units) appeared at 4.83-5.00ppm [24,44].Regarding product 5, the new peak at  2.35-2.50ppm represents the resonance of the two methylene groups of cyclohex-3-enecarboxylic acid; however the singlet peak at  5.74-5.82represents the two hydrogen protons of cyclohex-3-enecarboxylic acid and the new peak at  11.50 ppm originated from the proton of COOH of cyclohex-3-enecarboxylic acid substituent (Figure 2(b)) confirming again the successful grafting of this substituent onto chitosan molecule at the amino group.
The DDA was calculated to be 91% in chitosan (Table 1) from the integral ratio between the proton on the C-2 and the GlcN unit protons.However, DDA of N-(6-carboxyl cyclohex-3-ene carbonyl) chitosans ranged from 0.54 to 0.84.Determination of the DS value was based on the ratio between the areas of the protons in the substituent and the protons of the pyranose unit.The result proved that increasing the DS resulting in a decrease of the DDA that confirms the reaction mainly occurred on the amino group on C-2 of the GlcN.FW was calculated as follows: FW = 161 × DDA + 203 × DA + MW of N-(6-carboxyl cyclohex-3-ene carbonyl) glucosamine unit × DS, where 161 and 203 are the FW of GlcN unit and GlcNAc, respectively.The results indicate that the FW was increased with the mol ratio increase.FW for chitosan, 1, 2, 3, 4, and 5 were 177.27,191.58,203.63, 210.14, and 224.70, respectively, and the average molecular weight of the compounds ranged from 3.87 to 4.91 × 10 5 g/mol (Table 1).The derivatives were reported to have average MW 3.01, 3.39, 3.87, 4.11, and 4.77 × 10 5 Da compared to 2.76 × 10 5 for chitosan indicating that the MW is correlated with the intrinsic viscosity ().
Further evidence for confirmation the chemical structure was obtained from 13 C-NMR spectroscopy.The carbon peaks due to C=O and CH 3 were found at 174.97 and 22.08 ppm in chitosan, respectively.When we grafted THPA into the amino group of chitosan, new chemical shifts at 176. 23 2. The data shows that the derivatives were more soluble than chitosan in water and diluted aqueous acetic acid solutions.In addition, the derivatives became soluble also in alkaline basic medium (sodium hydroxide solution) indicating the presence of the carboxyl group in the product [33].

Antibacterial Activity of Chitosan Derivatives.
The in vitro antibacterial activities of chitosan and N-(6-carboxyl cyclohex-3-ene carbonyl) chitosans against E. carotovora, R. solanacearum, R. fascians, and R. radiobacter are presented in Table 3 as MIC.The results proved that the a MIC is a minimum inhibitory concentration.MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation.
derivatives showed higher inhibition (MIC ranged from 240 to 1125 mg/L) than chitosan (MIC ranged from 1050 to 1880 mg/L) against all the tested bacteria.The data indicated that the inhibitory effects increased with the DS increase and the most inhibition effect was observed with compound 5 (DS = 0.40) with MIC of 510, 735, 240, and 385 mg/L against E. carotovora, R. solanacearum, R. fascians, and R. radiobacter, respectively.
Recently, the antibacterial activity of N-(4-carboxybutyroyl) chitosan derivatives at different DS values (0.10, 0.25, 0.48 and 0.53) was evaluated against Agrobacterium tumefaciens and E. carotovora.They reported that the compound of DS 0.53 was the most active one (MIC = 725 and 800 mg/L against E. carotovora and A. tumefaciens, resp.)[33].Previous studies on the antibacterial activity of N,N,N-dimethylalkyl chitosans as water-soluble derivatives and N-(benzyl) chitosan derivatives was done under the same conditions as the present study against E. carotovora and A. tumefaciens [41,45].

Antifungal Activity of Chitosan Derivatives.
The antifungal activity of chitosan and N-(6-carboxyl cyclohex-3ene carbonyl) chitosan derivatives against A. alternata, B. cinerea, Bd. theobromae, F. oxysporum, F. solani, P. digitatum, Ph. infestans, and S. sclerotiorum by using mycelia radial growth technique is presented in Table 4.The native chitosan molecule in the present study has low activity against the tested pathogens.Conversely, N-(6-carboxyl cyclohex-3-ene carbonyl) chitosan derivatives appeared to be synergistic, and a marked increase in antifungal activity was noted.Unmodified chitosan showed EC 50 higher than 3000 mg/L against B. cinerea, Bd. theobromae, P. digitatum, and S. sclerotiorum.However, a notable effect was exerted against A. alternata, F. oxysporum, F. solani, and P. infestans with EC 50 of 2849, 2697, 1848, and 1600 mg/L, respectively.The derivatives were more active than chitosan and the inhibition effect was increased significantly with the DS increase.Compound 5 with a DS 0.40 exerted the prominent antifungal activity with EC 50 of 683, 774, 501, 500, 260, 417, 298, and 763 mg/L against A. alternata, B. cinerea, Bd. theobromae, F. oxysporum, F. solani, P. digitatum, Ph. infestans, and S. sclerotiorum, respectively.However, compound 1 with the lowest DS (0.09) was the lowest active one with EC 50 of 1390, 1560, 971, 1338, 641, 1645, 511, and 1746 mg/L, respectively.In regard to the susceptibility of the eight tested fungi, it can be noticed that the fungi of F. solani and Ph.infestans were more susceptible (EC 50 ranged from 260 to 641 mg/L) to these derivatives than the other tested fungi.
The present study noticed that the antifungal activity was increased dramatically with an increase in DS and MW values.This phenomenon is in agreement with Zhong and others who reported that the amount of grafted acetyl phenyl-thiosemicarbazone in the acetyl and benzoyl phenylthiosemicarbazone derivatives of chitosan correlated with the antifungal activity of the derivatives [46,47].Jia and coauthors found that pyridine chitosan exhibited enhanced antifungal activity by comparison with pristine chitosan [48].The values of the MIC and the minimal fungicidal concentration of pyridine chitosan against B. cinerea were 130 and 4000 mg/L, respectively.Severe morphological changes of pyridine chitosan-treated B. cinerea were observed, indicative of the fact that pyridine chitosan could damage and deform  [24] against B. cinerea (EC 50 = 430, 440, 450, and 500 mg/L, resp.) and were 12-to 13-fold more active than chitosan.However, N-(benzo[d] [1,3]dioxol-5-ylmethyl) chitosan and N-(methyl-4H-chromen-4-one) chitosan were the most active within five heterocyclic chitosan derivatives against P. debaryanum and F. oxysporum [18].
The most antifungal mechanism of chitosan is that the positively charged chitosan gives this one great physiological and biological polymer property.It consumes electronegative charges on the surface of the microbial cells resulting in change in cell wall permeability, so that the interaction results in leakage of electrolytes, intracellular protein components, and the disturbance of the plasma membrane [21,50,51].In addition, chitosan caused a decrease in the H + -ATPase activity on plasma membrane of R. stolonifer; this effect could provoke the accumulation of protons inside the cell, which would result in the inhibition of the chemiosmotic driven transport that allows the H + /K + exchange [52].Another important mechanism on fungi stated that the chitosan enters fungal cells and then adsorbs essential nutrients, which inhibit or retard the synthesis of mRNA and protein [53].Chitosan also acts as a chelating agent that selectively binds trace metals and thus inhibits toxin production and microbial growth [54].

Conclusion
The antimicrobial activity is one of the most important bioactivities of chitosan and it will be improved in some of the derivatives, which is determined by the groups grafted to chitosan.In the present study, N-(6-carboxyl cyclohex-3ene carbonyl) chitosans with five DS values were synthesized and tested against some plant pathogens.The products at the tested concentrations exhibited a wide range of the antibacterial and antifungal activity in vitro.It was observed that the highest DS value is more active than the lowest one.Therefore, we can suggest that such products could be used in plant protection program for controlling the plant pathogens that cause destruction of the crops and vegetables.However, formulating of such compounds is essential for commercial uses of the pesticidal chitosan products with further in vivo studies being essentially needed.Such formulations can be used in organic and conventional agricultural systems if the formulations are improved for foliar application.

Figure 1 :
Figure 1: Reaction scheme of chitosan with tetrahydrophthalic anhydride at different mol ratios.
, 177.89, and 178.23 ppm appeared due to the resonance of the carbonyl group (C=O) present in the carboxyl group (COOH) and