Synthesis, characterization, and antifungal activity of novel quaternary chitosan derivatives

Article abstract of DOI:10.1016/j.carres.2010.05.029

Three novel quaternary chitosan derivatives were successfully synthesized by reaction of chloracetyl chitosan (CACS) with pyridine (PACS), 4-(5-chloro-2-hydroxybenzylideneamino)-pyridine (CHPACS), and 4-(5-bromo-2-hydroxybenzylideneamino)-pyridine (BHPACS

Full text of DOI:10.1016/j.carres.2010.05.029

Carbohydrate Research 345 (2010) 1896–1900
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Carbohydrate Research
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Synthesis, characterization, and antifungal activity of novel quaternary
chitosan derivatives
c
Rongchun Li b,c, Zhanyong Guo a, , Pingan Jiang
*
a Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China
b Department of Chemistry, Dezhou University, Dezhou 253023, China
c College of Pratacultural and Environmental Science, Xinjiang Agriculture University, Urumqi 830052, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three novel quaternary chitosan derivatives were successfully synthesized by reaction of chloracetyl
chitosan (CACS) with pyridine (PACS), 4-(5-chloro-2-hydroxybenzylideneamino)-pyridine (CHPACS),
and 4-(5-bromo-2-hydroxybenzylideneamino)-pyridine (BHPACS). The chemical structure of the pre-
pared chitosan derivatives was confirmed by Fourier transform infrared (FT-IR) and 13C nuclear magnetic
resonance (13C NMR) and their antifungal activity against Cladosporium cucumerinum, Monilinia fructicola,
Colletotrichum lagenarium, and Fusarium oxysporum was assessed. Comparing with the antifungal activity
of chitosan, CACS, and PACS, CHPACS and BHPACS exhibited obviously better inhibitory effects, which
should be related to the synergistic reaction of chitosan itself with the grafted 2-[4-(5-chloro-2-hydrox-
ybenzylideneamino)-pyridyl]acetyl and 2-[4-(5-bromo-2-hydroxybenzylideneamino)-pyridyl]acetyl.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 23 January 2010
Received in revised form 24 May 2010
Accepted 31 May 2010
Available online 4 June 2010
Keywords:
Quaternary chitosan
Chloracetyl chitosan
5-Chlorosalicylaldehyde
5-Bromosalicylaldehyde
Antifungal activity
1. Introduction
fructicola, Colletotrichum lagenarium, and Fusarium oxysporum that
employed the method of Guo.14 Besides, the application of chitosan
Chitosan has attracted people’s attention for its physiochemical
characteristics and its bioactivities.1–6 The antimicrobial properties
of chitosan and its derivatives have been widely explored.7–9 To
improve the antimicrobial activity of chitosan, researchers have
prepared many derivatives which showed higher antimicrobial
activities than chitosan, but the antimicrobial activities of these
materials are still lower than those of the antimicrobial agents cur-
rently used.10–15
is limited for its poor solubility in water or high pH region. There-
fore, taking low molecular weight chitosan with high solubility
and low viscosity in water at physiologically acceptable pH values
as starting material can enlarge the scope of application. In this pa-
per, we used water-soluble low molecular chitosan as starting
material. The derivatives were expected to have better solubility
and antifungal activity.
N,O-Acyl chitosan derivatives were synthesized and their fungi-
cidal activities against plant fungus were examined. The derivatives
were more active against the tested plant pathogens than chitosan
itself.16 In addition, many reports have demonstrated that aromatic
nucleus Schiff bases have good bioactivities.17,18 It is also reported
that Salicylaldehyde derivatives, with one or more halo-atoms in
the aromatic ring, showed a variety of biological activities.19 We
describe here the preparation of three novel derivatives of chitosan
including the above active groups, (2-pyridyl)acetyl chitosan chlo-
ride (PACS), 2-[4-(5-chloro-2-hydroxybenzylideneamino)-pyridyl]
acetyl chitosan chloride (CHPACS), and 2-[4-(5-bromo-2-hydroxyb-
enzylideneamino)-pyridyl]acetyl chitosan chloride (BHPACS)
(Scheme 1), as well as their antifungal activity against four plant-
threatening pathogenic fungi Cladosporium cucumerinum, Monilinia
2. Materials and methods
2.1. Materials
Chitosan was purchased from Qingdao Baicheng biochemical
Corp. (China). Its degree of deacetylation was 97% and the viscos-
ity-average molecular weight was 2.0 ꢀ 105. The water-soluble
chitosan was prepared in our laboratory by the method of hydro-
gen peroxide (H2O2) hydrolysis and the viscosity-average molecu-
lar weight was 7.0 ꢀ 103. The other reagents were of analytical
grade and used without further purification.
2.2. Analytical methods
The Fourier transform infrared (FT-IR) spectrum was measured
in KBr pellets with a JASCO FT-IR-4100 instrument. The 13C nuclear
magnetic resonance (13C NMR) spectrum was measured with a
* Corresponding author. Tel.: +86 535 2109171; fax: +86 535 2109000.
E-mail address: qdioqdio@yahoo.com.cn (Z. Guo).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.05.029

R. Li et al. / Carbohydrate Research 345 (2010) 1896–1900
1897
Scheme 1. Synthetic route of CACS, PACS, CHPACS, and BHPACS.
Bruker AVIII-500 spectrometer. PACS had well water solubility, but
CHPACS and BHPACS had slight water solubility. So the 13C NMR
was carried out in D2O (PACS) and in DMSO (CHPACS and BHPACS).
2.3. The synthesis of the derivatives of chitosan
The synthesis of 5-chlorosalicylaldehyde and 5-bromosalicylal-
dehyde was, respectively, carried out according to the procedure of
Liu et al.20 and Liang and Chin.21 The chloracetyl chitosan (CACS)
was synthesized referring to the procedure of Hu et al.22 1.61 g
chitosan was dispersed in 100 mL N-methyl-2-pyrrolidone (NMP)
and stirred for 12 h at room temperature (rt). Then 0.02 mol chlo-
racetyl chloride was added. After stirring for 24 h at rt, the solution
was precipitated in ether and the precipitate was washed with
methanol and ether by turns and lyophilized. The synthesis of
PACS: a solution of 0.3 g CACS and 0.5 mL pyridine in 20 mL di-
methyl sulphoxide (DMSO) was stirred for 24 h at 60 °C. The solu-
tion was collected by precipitation with excess acetone and the
product was lyophilized, PACS was obtained. CHPACS and BHPACS
were synthesized as follows: a solution of 0.01 mol 5-chlorosalicyl-
aldehyde or 5-bromosalicylaldehyde and equimolar amount of 4-
amino-pyridine were refluxed in 30 mL ethanol for 4 h. Then the
solvent was evaporated under reduced pressure and the product
was dissolved by adding 20 mL DMSO. After the product dissolved
under stirring, 0.3 g CACS was added. The solution was stirred at
60 °C for 24 h and then precipitated with excess acetone and the
precipitate was filtered. The unreacted aldehyde, amine, and other
outgrowth were extracted in a Soxhlet apparatus with ethanol for
two days. The product was lyophilized.
2.4. Antifungal assays
Antifungal assays were performed based on the method of Guo
et al.14 Briefly, the compounds were dispersed in distilled water at
a concentration of 5 mg/mL. Then, each samples (chitosan, CACS,
PACS, CHPACS, and BHPACS) solution was added to sterilized pota-
to dextrose agar to give a final concentration of 100, 500, and
Figure 1. FT-IR spectra of chitosan, CACS, PACS, CHPACS, and BHPACS.
experiment was performed three times, and the data were aver-
aged. The Scheffe method was used to evaluate the differences in
antifungal index in antifungal tests. Results with P <0.05 were con-
sidered statistically significant.23
1000 l
g/mLꢁ1. After the mixture was cooled, the mycelium of fun-
gi was transferred to the test plate and incubated at 27 °C for 2–
3 days. When the mycelium of fungi reached the edges of the con-
trol plate (without the presence of samples), the antifungal index
was calculated as follows:
3. Results and discussion
3.1. Structure of the chitosan derivatives
Antifungal index ð%Þ ¼ ð1 ꢁ Da=DbÞ ꢀ 100;
where Da is the diameter of the growth zone in the test plates and
Db is the diameter of the growth zone in the control plate. Each
Figure 1 presents the comparison of the transmission FT-IR spec-
tra for CACS, PACS, CHPACS, and BHPACS with the original chitosan.

1898
R. Li et al. / Carbohydrate Research 345 (2010) 1896–1900
As far as the FT-IR spectra of CACS and chitosan are concerned, the
characteristic absorbance of –NH2 at 1600 cmꢁ1 disappeared. The
stretching vibration of the carbonyl groups C@O of acylamide at
1666 cmꢁ1 and of the ester group at 1750 cmꢁ1 could be obviously
observed.24,25 New peaks at 787 cmꢁ1 were assigned to the C–Cl
group. It indicated that CACS was obtained by the acylated reaction
between chloracetyl chloride and the groups (–NH2, –OH) of chito-
san. After pyridine grafted onto the CACS, the characteristic absor-
bance of C–Cl at 787 cmꢁ1 disappeared and new peaks at 1496,
779, and 3081 cmꢁ1 were due to the characteristic absorbance of
pyridine26 appeared in the spectra of PACS. In the spectrum of CHP-
ACS, new peaks appeared at 1481 cmꢁ1, 3135 cmꢁ1 which were
attributed to aromatic ring stretching vibrations and C–H stretching
vibrations, respectively. The out-of-plane pyridine deformation
vibrations were found at 767 cmꢁ1 and the peak at 829 cmꢁ1 was
due to benzene bending. Another prominent peak at 1284 cmꢁ1
was assigned to the C–O of phenolic hydroxyl group.26,27 The charac-
teristic absorbance of C–Cl at 787 cmꢁ1 disappeared and the peaks of
carbonyl groups of acylamide and ester were bathochromic shifted
to 1658 and 1712 cmꢁ1, respectively, due to the graft of the 4-(5-
chloro-2-hydroxybenzylideneamino)-pyridine.28 For the similar
reason, the C–Cl characteristic absorbance at 787 cmꢁ1 disappeared
in the FT-IR spectra of BHPACS. And both the appearing of the new
peaks at 1477 cmꢁ1, 1284 cmꢁ1, 829 cmꢁ1, 767 cmꢁ1 and the car-
bonyl groups of acylamide and ester bathochromic shifting to
1658 and 1712 cmꢁ1 indicated the successful synthesis of BHPACS.
The 13C NMR spectrum of PACS, CHPACS, and BHPACS (Fig. 2)
further confirmed the success of the preparation.
127.8 ppm (pyridine ring carbons).29 d 55.8–99.7 ppm (pyranose
rings), d 60.1, 38.3 ppm (methylene carbon of –OCOCH2– and
–NH2COCH2–).30
Spectral data for CHPACS. 13C NMR/DMSO: d 166.1, 166.6 ppm
(C@O of ester group and acylamide).25 d 159.3, 144.5, 109.3 ppm
(pyridine ring carbons), d 159.3 ppm (carbon of N@CH–), d 118.9,
120.4, 122.6, 131.0, 132.6, 159.3 ppm (benzene ring carbons).31
d
58.6–101.1 ppm (pyranose rings). The peak of methylene carbon
in –OCOCH2– was included in the peaks at about 60 ppm and that
of methylene carbon in –NH2COCH2– was covered by peaks of
DMSO.30
Spectral data for BHPACS. 13C NMR/DMSO: d 166.0, 166.5 ppm
(C@O of ester group and acylamide).25 d 159.4, 144.5, 109.6 ppm
(pyridine ring carbons), d 159.4 (carbon of N@CH–), d 119.1–
135.3, and 159.4 ppm (benzene ring carbons).31 d 58.8–100.9
ppm (pyranose rings). The chemical shift of methylene carbon in
–OCOCH2– was included in the peaks at about 60 ppm and that
of methylene carbon in –NH2COCH2– was covered by peaks of
DMSO.30
3.2. Solubility of the chitosan derivatives
The water solubility of chitosan and the derivatives is listed in
Table 1. Because of the decrease of hydrophilic hydroxyl and amino
groups, CACS exhibited soft water solubility. After pyridine substi-
tuted the chlorine in CACS, PACS was obtained and quaternary
ammonium salt structure was formed. Therefore, PACS has favor-
able water solubility. The solubility of CHPACS and BHPACS was
less than that of material chitosan, maybe due to the hydrophobic
moiety at the end of the molecular chains.
Spectral data for PACS. 13C NMR/H2O: d 173.1 ppm (C@O of es-
ter group), 166.0 ppm (C@O of acylamide).25 d 146.8, 145.5 and
Figure 2. 13C NMR spectra of PACS, CHPACS, and BHPACS.

R. Li et al. / Carbohydrate Research 345 (2010) 1896–1900
1899
Table 1
Water solubility of CACS, PACS, CHPACS, and BHPACS
Compounds
Water solubility (%)
Test time (min)
Chitosan
CACS
PACS
CHPACS
BHPACS
30.5
1.5
25.5
6.5
5
20
5
10
10
6.0
Figure 4. The antifungal activities of chitosan, CACS, PACS, CHPACS, and BHPACS
against M. fructicola.
Figure 3. The antifungal activities of chitosan, CACS, PACS, CHPACS, and BHPACS
against C. cucumerinum.
3.3. Antifungal activity
The most economically important seed or plant diseases are
caused by the action of fungi. The need to find eco-friendly treat-
ments to avoid the use of aggressive chemicals that cause contam-
ination is growing. Here, we tested the antifungal activities of the
starting chitosan, CACS, PACS, CHPACS, and BHPACS against four
kinds of plant fungus. The results are demonstrated as follows.
Figure 5. The antifungal activities of chitosan, CACS, PACS, CHPACS, and BHPACS
against C. lagenarium.
3.3.1. Antifungal activities of chitosan, CACS, PACS, CHPACS, and
BHPACS against C. cucumerinum
As shown in Figure 3, chitosan and its derivatives had antifungal
activities at all the tested concentrations against C. cucumerinum.
CACS showed better antifungal activity than chitosan at 1000 lg/
mL with the inhibitory index 41.4%. The inhibitory indices of PACS,
3.3.3. Antifungal activities of chitosan, CACS, PACS, CHPACS, and
BHPACS against C. lagenarium
Figure 5 shows the antifungal activity against C. lagenarium of
chitosan, CACS, and all the derivatives. All the samples have anti-
fungal activity against C. lagenarium. And the inhibitory indices of
all the samples mounted up with increasing concentration. The
inhibitory indices of chitosan, CACS, PACS, CHPACS, and BHPACS
CHPACS, and BHPACS enhanced with increasing concentration.
With the inhibitory indices 100% at 500 lg/mL, CHPACS and BHP-
ACS exhibited much better antifungal activities than chitosan and
CACS. However, the antifungal activity of PACS against C. cucumer-
inum did not reveal apparent improvement than chitosan and
CACS.
at 500
tively, and 36.6%, 36.6%, 30.2%, 71.0%, and 75.8%, respectively, at
1000 g/mL. It indicated that the antifungal activities of CHPACS
and BHPACS against C. lagenarium were markedly better than those
of chitosan, CACS, and PACS. The results, again, suggested that 5-
chloro-2-hydroxylbenzylideneamino and 5-bromo-2-hydroxyben-
zylideneamino groups maybe antifungal function groups.
lg/mL were 32.0%, 26.6%, 15.3%, 45.3%, and 63.3%, respec-
l
3.3.2. Antifungal activities of chitosan, CACS, PACS, CHPACS, and
BHPACS against M. fructicola
As shown in Figure 4, chitosan, CACS, and PACS had soft anti-
fungal activities against M. fructicola just as C. cucumerinum was
concerned. The inhibitory indices of chitosan, CACS, PACS, CHPACS,
3.3.4. Antifungal activities of chitosan, CACS, PACS, CHPACS, and
BHPACS against F. oxysporum
and BHPACS at 500
respectively. The inhibitory indices of chitosan, CACS, PACS, CHP-
ACS, and BHPACS at 1000 g/mL were 9.3%, 14%, 21.5%, 100%,
and 100%, respectively. It indicated that CHPACS and BHPACS have
potent activities. Comparing the difference of the compounds
structure, we can speculate that the increased antifungal activities
of CHPACS and BHPACS may benefit from 5-chloro-2-hydroxylben-
zylideneamino and 5-bromo-2-hydroxylbenzylideneamino groups
in the compounds.
l
g/mL were 6.9%, 14%, 19.1%, 100%, and 100%,
F. oxysporum, also referred to as Panama disease or Agent
Green, is a fungus that causes Fusarium wilt disease in more than
a hundred species of plants. It does so by colonizing the water-
conducting vessels (xylem) of the plant. As a result of this block-
age and breakdown of xylem, symptoms such as leaf wilting, yel-
lowing, and eventually plant death appear in plants. Therefore,
the study on antifungal agents is significative. The fungicidal
activities of the compounds toward F. oxysporum are depicted in
Figure 6. As shown in Figure 6, the diversification trend of the
l

1900
R. Li et al. / Carbohydrate Research 345 (2010) 1896–1900
the environmental issue. So, this kind of chitosan derivatives and
similar compounds are worthy of further studying. We have been
studying this series of compounds more extensively. The work will
be demonstrated in the near future.
Acknowledgments
This work was supported by the Knowledge Innovation Pro-
gram of the Chinese Academy of Sciences, Grant No. kzcx2-yw-
225, which is gratefully acknowledged.
Thanks for the financial support by the foundation of Special
Prize of President Scholarship for Postgraduate Students of Chinese
Academy of Sciences.
References and notes
Figure 6. The antifungal activities of chitosan, CACS, PACS, CHPACS, and BHPACS
against F. oxysporum.
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l
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4. Conclusion
In this work, three novel quaternized chitosan derivatives were
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