Synthesis and characterization of new chitosan-based Schiff base compounds

Article abstract of DOI:10.1016/j.carbpol.2016.02.076

Chitin (Cn) was extracted from the armors of crustaceans Astacus leptodactylus (Lake Sevan, Armenia) and then converted to chitosan (Cs), its deacetylated derivative. Novel Schiff bases (CsSB) were synthesized by interaction of Cs with 4-(2-chloroethyl)be

Full text of DOI:10.1016/j.carbpol.2016.02.076

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Title: Synthesis and characterization of new chitosan-based
Schiff base compounds
Author: Vasak B. Gavalyan
PII:
S0144-8617(16)30141-2
DOI:
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http://dx.doi.org/doi:10.1016/j.carbpol.2016.02.076
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23-2-2016
28-2-2016
Please cite this article as: & Gavalyan, Vasak B., Synthesis and characterization
of new chitosan-based Schiff base compounds.Carbohydrate Polymers
http://dx.doi.org/10.1016/j.carbpol.2016.02.076
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Synthesis and characterization of new chitosan-based Schiff base compounds1
Vasak B. Gavalyan
AANL Foundation (Yerevan Physics Institute)
2-Alikhanyan Brothers St., Yerevan 0036, Armenia
E-mail address: vasak@mail.yerphi.am
Tel.: 093–02–13–76
Highlights
 New chitosan Schiff bases are synthesized.
 4-(2-chloroethyl) and 4-(2-bromoethyl)benzaldehydes are used in the synthesis.
 The product composition slightly depends on the reaction temperature (room temperature vs
80C).
 Vinyl derivatives of chitosan Schiff base are obtained in alkaline environment.
A B S T R A C T
Chitin (Cn) was extracted from the armors of crustaceans Astacus leptodactylus (Lake Sevan,
Armenia) and then converted to chitosan (Cs), its deacetylated derivative. Novel Schiff bases
(CsSB) were synthesized by interaction of Cs with 4-(2-chloroethyl)benzaldehyde (aldehyde-1)
and 4-(2-bromoethyl)benzaldehyde (aldehyde-2), and underwent dehydrohalogenation, under
basic conditions (10°C), to yield respective vinyl derivatives. All newly synthesized compounds
were structurally characterized by solubility tests, elemental analysis, infrared spectroscopy
(FTIR), thermogravimetry (TGA), proton nuclear magnetic resonance (1H NMR), and X-ray
diffraction (XRD).
1 Abbreviations:
Cn, chitin; Cs, chitosan; CsSB, chitosan Schiff base.;Aldehyde-1, 4-(2-
chloro)ethylbenzaldehyde.;Aldehyde-2, 4-(2-bromo)ethylbenzaldehyde;FTIR spectroscopy,
Fourier-Transformed Infrared spectroscopy;TGA, thermogravimetry;1H NMR, proton nuclear
magnetic resonance;XRD, X-ray diffraction;DRON-3, general purpose X-ray diffractometer-
3;DS, degree of substitution;ppm, parts per million;CsSBRTCl, CsSB from aldehyde-1
synthesized at RT;CsSB80Cl, CsSB from aldehyde-1 synthesized at 80C;CsSBRTBr, CsSB
from aldehyde-2 synthesized at RT.;CsSB80Br, CsSB from aldehyde-2 synthesized at
80C.;CsSBVinyl-1, vinyl derivative from CsSBRTCl;CsSBVinyl -2, vinyl derivative from

CsSBRTBr.;TG, thermogravimetric;DTG, derivative thermogravimetric;DTA, differential
thermal analysis;X ,halogen.
Keywords
Chitin, chitosan, 4-(2-haloethyl)benzaldehyde, new chitosan Schiff base
1. Introduction
Chitosan (Cs) [β-(1→4)-2-amino-2-deoxy-D-glucopyranose] is the product of deacetylation
of chitin (Cn) [β-(1→4)-2-acetamido-2-deoxy-D-glucopyranоse]. Some products of Cs chemical
transformations are biocompatible, biodegradable and non-toxic; they exhibit antioxidant
properties and have been widely used in biomedicine (Kumirska, Weinhold, Thöming, &
Stepnowski, 2011), environmental protection (Kocak, Sahin, Arslan, & Ucan, 2012), agriculture
(Hadrami, Adam, Hadrami, & Daayf, 2010), and textile industry (Klaykruayat, Siralertmukul, &
Srikulkit, 2010). Numerous derivatives of Cs were synthesized due to the pending amino and
hydroxyl groups peripherially attached to the macromolecule backbone (Santos, Dockal,
Cavalheiro, 2005; Sajomsang, Tantayanon, Tangpasuthadol, Thatte, & Daly, 2008; Mourya,
Inamdar, & Tiwari, 2010). The Schiff bases that can be obtained by interaction of the Cs amino
groups with aldehydes and ketones are of immediate interest as biomaterials endowed with a set
of unique properties (Jiao et al., 2011; Sashiva & Aiba, 2004). In particular, the newly formed
imino groups increase the physiological activity of chitosan Schiff bases (CsSB), so that they can
act as antibacterial (Soliman, El-Kousy, Abd-Elbary, & Abou-zeid, 2013), antitumor (Park,
Chung, Choi, & Park, 2011), antimicrobial and anticorrosive (Mohamed & Fekry, 2011) agents.
CsSB were also synthesized with aromatic aldehydes as carbonyl components (Santos, Dockal,
& Cavalheiro, 2005; Sajomsang, Tantayanon, Tangpasuthadol, Thatte, & Daly, 2008; Jiao et al.,
2011). The existence of electron-donating and electron-withdrawing substituents at the aromatic
ring exerted a noticeable influence on the electrophilicity of the carbonyl group and on the degree
of substitution (DS) of CsSB. As a carbonyl compound, to the best of our knowledge, 4-(2-
chloroethyl) benzaldehyde (aldehyde-1) and 4-(2-bromoethyl) benzaldehyde (aldehyde-2) have
not been used in the CsSB synthesis. Structurally, an attractive feature in aldehydes chosen is the
presence of a XCH2CH2-group that allows for elimination reaction and for introducing an
unsaturation in the select positions of the molecular assembly. In particular, pending vinyl groups
would allow for the synthesis of new polymeric systems with potentially valuable properties.
Herein we report on the synthesis of new CsSB derivatives and their structural characterization
by means of TGA, 1H NMR, FTIR, and XRD as well as elemental analysis.

2 Experimental
2.1. Materials and methods
Reagents and solvents were purchased from various vendors and used without further
purification. Sodium hydroxide, potassium hydroxide, hydrochloric acid, sulfuric acid, glacial
acetic acid, acetone, diethyl ether, methanol, ethanol, benzene, chloroform, dichloromethane,
tetrahydrofuran, N,N-dimethylformamide, silver nitrate, and dimethyl sulfoxide were purchased
from Reahim (chemically pure), tetrachloromethane and trifluoroacetic acid were delivered by
Sigma-Aldrich, and sodium bicarbonate, hexamethylentetramine, magnesium sulfate, aqueous
ammonia, and sodium borohydride were obtained from Reahim (pure for analysis). Deiterium
oxide, trifluoroacetic acid-d, and dimethyl sulfoxide-d6 were acquired from Cambrige Isotope
Laboratories (Andover, MA). In the solubility tests, the ratio of the product mass (mg) and a
volume of solvent (mL) was equal to 1 : 2. Elemental analyses (C, H, N) were performed on Euro
EA 3000 analyzer (Italy). Melting temperatures were measured using a Boetius tipe microscope
with a heating stage. Attenuated total reflection Fourier-transform infrared spectra (FTIR ATR)
were registered by Nicolet 5700 spectrometer (ZnSe prism, Happ-Genzel apodization, correction
of ATR distortion, number of scans 32, resolution 4 cm-1). The thermogravimetry (TGA) was
carried out in air atmosphere using Derivatograph Q-1500 (Hungary; temperature gradient: room
temperature to 700oC / 10oC/min). The initial weight of each sample was 100mg. The proton
nuclear magnetic resonance (1H NMR) spectra were measured using Varian Mercury 300VX
300MHz spectrometer at 303K. X-ray diffraction (XRD) measurements were conducted using
DRON-3 X-ray diffractometer with CuKα radiation (2θ, 10o to 80o).
2.2.Cn extraction
Armors of crayfish Astacus leptodactylus (lake Sevan, Armenia) were cleaned of pectins,
grinded to a fine powder, and extracted according to the literature protocol (Lertsutthiwong,
How, Chandrkrachang, & Stevens, 2002; "Process 2"). Demineralization was carried out with
dilute HCl (3.5% HCl; powder mass to acid volume (w/v) was equal to 1:14) at RT for 2.5h.
Under continuous stirring, the acid solution was added portionwise until the release of carbon
dioxide ceased off completely. The product was washed with distilled water until no chloride
ions remained in the solution (AgNO3 test), then dried at 60C for 1.5h. Deproteinization was
conducted with aq. NaOH (5%; w/v, 1:15) at 80C for 5h. The product was then washed with
distilled water until neutral reaction (pH=7), dried at 65C for 12h, and decolorized in the Soxlet
apparatus by refluxing a mixture of benzene and ethyl alcohol (v/v, 1:1) for 44h. The isolated
product was then dried at 65C for 22h to yield pure Cn (28%). Elemental analysis, %: C, 46.33;
N, 6.98; H, 6.36; ash, 0.08. The degree of acetylation (DA) of Cn (90.5%) was calculated by
using elemental analysis data and an equation reported earlier (Kasaai, Arul, & Charlet, 2000).
2.3. Cs synthesis
Under nitrogen atmosphere, deacetylation of Cn (7g) was carried out with aq. NaOH (50%;
w/v, 1:14) and NaBH4 (w/w, 10:1) at 117÷120C for 2.5h. The product was washed with distilled

H2O until neutral reaction (pH=7), then rinsed with ethyl alcohol (x3), acetone (x3), and dried in
vacuum (10-1 torr; RT, 30h) to yield Cs (5.7g). Elemental analysis, %: C, 45.39; N, 7.70; H, 6.58;
ash, 0.2. DA of synthesized Cs (47.6%) was calculated by using elemental analysis data and an
equation reported earlier (Kasaai, Arul, & Charlet, 2000).
2.4. Cs purification
Cs (2.6g) was dissolved in diluted acetic acid (0.19M; 260ml H2O + 3ml CH3COOH) by
continuously stirring the resulting suspension for 6.5 h. The mixture was then neutralized with
NH4OH (10%; 675 ml, 2.85М; 1.5 h) and treated according to the literature protocol (Santos,
Dockal, & Cavalheiro, 2005). The product was washed with distilled water until neutral reaction
(pH=7) and dried in vacuum (10-1 torr; RT; 5.5h) to yield Cs-pure (2.2g).
2.5. Synthesis of aromatic aldehydes
4-(2-Chloroethyl)benzaldehyde (aldehyde-1) and 4-(2-bromoethyl)benzaldehyde (aldehyde-
2) were synthesized by method discrabed in (Pogosyan & Matsoyan, 1983).
A
solution of 4-(2-chloroethyl) benzyl chloride (18.9g, 0.1mol) and
hexamethylenetetramine (15.5g, 0.11mol) in dry chloroform (150mL) was heated for 2h at 70°C.
The reaction mixture was concentrated by 2/3 of the initial volume and diluted with ether (90ml).
The precipitate was filtered off, washed with ether, and refluxed in acetic acid (8M; 90mL) for
2h. Hydrochloric acid was added (10ml; 10M) and the reaction mixture was refluxed for another
5min. The crude mixture was extracted with ether, an organic layer was washed with water, then
with aqueous sodium bicarbonate solution, then again with water and dried over MgSO4. The
reaction mixture was fractionated by vacuum distillation (103-105°/2torr) to afford 4-(2-
chloroethyl) benzaldehyde (aldehyde-1) (5.15g, 31%), a colorless liquid. M.p. 32-33°C. 1H NMR
(300MHz, DMSO-d6/CCl4; 1/3, δ, ppm, J, Hz): 3.15 (2H, t, J=7.0, CH2), 3.80 (2H,t, J=7.0,
CH2Cl); 7.43-7.48 (2H, m) and 7.80-7.85 (2H, m, C6H4); 9.96 (1H.s, CHO).
4-(2-bromoethyl) benzaldehyde (aldehyde-2) was synthesized analogously (7.23g, 49%) by
1
using 4-(2-bromoethyl) benzylchloride as a reagent. A colorless liquid, m.p. 36-37°C. H NMR
(300MHz, DMSO-d6/CCl4; 1/3, δ, ppm, J, Hz): 3.15 (2H, t, J=7.0, CH2), 3.81 (2H, t, J=7.4,
CH2Br), 7.43-7.48 (2H, m) and 7.80-7.85 (2H, m, C6H4), 9.96 (1H.s, CHO).
2.6. Synthesis of chitosan Schiff base (CsSB)
2.6.1. Cs-pure (Section 2.4) in the amount of 0.42g (for the given DA of Cs of 47.6%, the
amount of 2-amino-2-deoxy-D-glucopyranose units in the initial polysaccharide sample was
0.22g, or 1.36mmol) was stirred in a solution of H2O (20mL) and CH3COOH (0.15mL, 0.15M)
at RT for 7h. A solution of aldehyde-1 (Section 2.5) (0.44g; 2.61mmol) in ethanol (5ml) was
added dropwise and the reaction mixture was stirred for additional 12h. When adding aldehyde
solution, the transparent and viscous (homogeneous) solution in the reactor gets cloudy, becomes
heterogeneous, and light yellow residual matter gradually precipitates. The precipitate was
filtered off, consecutively washed with acetone, ethanol and ether, and dried under reduced

pressure (10-1 torr, RT, 4h) to yield CsSBRTCl (0.46g, 9.4%). The same synthesis was carried
out at 80C by interaction of Cs-pure (Section 2.4) with aldehyde-1 (Section 2.5) to yield
CsSB80Cl (0.45g, 7.1%).
2.6.2. Cs-pure (0.83g; Section 2.4; for the given DA of Cs of 47.6%, the amount of 2-amino-
2-deoxy-D-glucopyranose units in the initial polysaccharide sample was 0.43g, or 2.7mmol) was
dissolved in a mixture of H2O (40mL) and CH3COOH (0.3ml, 0.15M) and stirred at RT for 7h. A
solution of aldehyde-2 (Section 2.5) (1.06g, 5.0mmol) in ethanol (10mL) was added dropwise,
and the reaction mixture was stirred for additional 12h. When adding aldehyde solution, the
transparent and viscous (homogeneous) solution in the reactor gets cloudy, becomes
heterogeneous, and tan residual matter gradually precipitates. The precipitate was filtered off,
consecutively washed with acetone, ethanol and ether, then dried in vacuum (10-1 torr, RT, 4h) to
yield CsSBRTBr (0.89g, 6.4%). The same synthesis was carried out at 80C by interaction of Cs-
pure (Section 2.4) with aldehyde-2 (Section 2.5) in the same quantities as described above. All
other procedures, parameters and conditions were identical. The product, CsSB80Br, was
obtained in an 8.3% yield (0.91g).
2.7. Preparation of vinyl derivatives of chitosan Schiff bases (CsSBVinyl): Poly-{ 4-hydroxy-5-[4-
hydroxy-6-hydroxymethyl-5-oxy-3-(4-vinylbenzylideneamino)tetrahydro-2H-pyran-2-yloxy]-6-
hydroxymethyl-2-oxytetrahydro-2H-pyran}
2.7.1. A solution of CsSBRTCl (0.26g; Section 2.6.1) in acetic acid (15mL; 0.15M) was stirred at
RT for 8h, and then added in small portions to an aqueous solution of NaOH (5N; 3.75g of
NaOH + 19mL of H2O) at 10C (30min). The precipitate was filtered, washed with distilled
water to the neutral reaction (pH=7), then rinsed consecutively with ethanol and acetone. Upon
drying under reduced pressure (10-1 torr; RT, 4h), the colorless CsSBVinyl-1 was isolated in a
yield of 64.8% (0.246g). CsSBVinyl-2 was synthesized analogously by using CsSBRTBr as a
substrate (0.244g, 89.8%).
3. Results and discussion
The scheme below shows some parameters of Cs interaction with aldehyde-1 and aldehyde-2
(CsSB formation, Reaction 1) and dehydrohalogenation of the new CsSB substrates (CsSBVinyl-
1 and CsSBVinyl-2 formation, Reaction 2). CsSB products are light-yellow, tan, or colorless
powders. In Table 1, the results of elemental analysis and yields of the synthesized products are
presented. The yields of the CsSB products were determined by the equation reported in
literature (Salama, Saad & Sabaa, 2015). Solubility of the synthesized products in various
solvents was investigated. In particular, it was established that said products do not dissolve in
such solvents as acetone, methanol, ethanol, diethyl ether, chloroform, dichloromethane,
benzene, tetrahydrofuran, N,N-dimethylformamide, and dilute sulfuric acid. In contrats, the
products dissolved in the mixtures of dimethylsulfoxide/trifluoroacetic acid (DMSO/TFA;1:1,
v/v), and partially dissolved, or swelled in other solvents (Table 2).
According to TGA data (see thermogravimetric curves TG in Fig.1, a), in temperature range
from 70oC to 140oC the mass loss of extracted Cn sample makes 6% of initial weight. We can
assume that the mass loss in question is mainly related to the release of adsorbed water. With the

temperature increase, from 140oC to 260oC, the sample loses another 2% of its mass. The more
intense thermal degradation lies within the temperature range of 261-420oC: the mass loss
reached 56.5%. According to the published data (Arora, Lai, Kumar, Kumar M, & Kumar,
2011), this thermal degradation is related to the isolation of the acetamide group, destruction
of the polysaccharide chain, and breakdown of the glucopyranose cycle. At about 420oC, slow
emission of volatile products, such as CO and CO2, and formation of the carbon residue begin.
These TG data are in a good agreement with those on shrimp Cn (Wanget et al., 2013) and raw
Cn (Khor, Wu, Lim, & Guo, 2011). Thermal behavior of the Cs (Fig.1, b) is somewhat different
from that of Cn. Here, in temperature range from 68oC to 124oC (see thermogravimetric curves
TG in Fig.1, b)mass loss makes up to 8.6% of the initial weight. The more intense thermal
degradation was observed at a point that is approximately 16oC lower than detected for Cn; it
covers the temperature range from 245oC to 340oC with the mass loss of 38.4%. According to the
data obtained by derivative thermogravimetry (DTG), the temperature minimum for the more
intense degradation is 300oC. The TG and DTG data show practically the same characteristics
for Cs (Fig.1, b) and Cs-pure samples (Fig.1, c). It is worthy to mention that the lower starting
point of thermal degradation, when converting Cn to Cs, can be explained by the fact that the
acetamide groups are partially substituted by the amine groups having inferior thermal stability
(Pawlak & Mucha, 2003); it can also be due to the depolymerization processes that could take
place during Cn deacetylation reaction. The TGA data for CsSBRTCl (a), CsSB80Cl (b) and
CsSBVinyl-1 (c) are depicted in Figure 2. It is worth mentioning that all three samples showed
the same thermal degradation pattern. According to the TGA data, the losses of initial mass for
CsSBRTCl, CsSB80Cl and CsSBVinyl-1 makes 4.2% at 60-117oC (Fig.2, a), 4.0% at 60-117oC
(Fig.2, b), and 1.7% at 60-97oC (Fig.2, c), correspondingly
On the TG curves for CsSBRTCl (184-254oC), CsSB80Cl (200-265oC) and CsSBVinyl-1
(200-261oC), the more intense mass loss makes 18.9%, 17.4% and 8.6%, respectively; the
minimums on DTG curves are at 221oC, 228oC and 230oC, correspondingly. At higher
temperatures up to 700oC thermal degradation of these three samples (Fig.2. a, b and c) presents
a smooth mass drop (TG curves) with the mass losses 39%, 45% and 34%, respectively. The
TGA parameters are in a good agreement with the literature reports (Tirkistani, 1998) indicating
that the thermal stability of CsSB is lower than that of Cs. It is probably due to the presence of
new functional groups in CsSB, which break down under the experimental conditions applied
(heating, oxygen-containing or neutral environment) and initiate further destruction of the
polysaccharide chain.
Due to the dehydrochlorination of CsSBRTCl and dehydrobromination of CsSBRTBr
(Reactions 1 and 2), new imine and vinyl groups are introduced into the polymeric chain. The
FTIR spectra of Cn (a), Cs (b) and Cs-pure (c) (Fig. 3) show characteristic absorptions of amide
1 (C=O) of the Cn acetamide group and splitting of this absorption band near 1619 cm-1 (C–N)
and 1660 cm-1 (C=O) (Fig.3, a). According to the literature data (Kumirska et al., 2010), these
are just the unique characteristics of α-polymorph Cn. The comparative analysis of the FTIR data
(Fig.3) shows that the spectra of Cs (b) and Cs-pure (c) are identical and substantially different
from the spectrum of Cn, i. e., an intensity of the absorption near 1660 cm-1 is decreased and a
new absorption characteristic for amino groups is detected at 1592 cm-1 (Paulino, Simionato,
Garcia, & Nozaki, 2006). This fact represents an evidence for Cn deacetylation and formation of
Cs. It should also be emphasized that the FTIR data (Fig. 3) are in a good agreement with Cn and
Cs spectra published previously (Brugnerotto et al., 2001). Figure 4 depicts FTIR spectra of
CsSBRTCl (a), CsSB80Cl (b) and CsSBVinyl-1 (c). These spectra are different from those in

Figure 3 (Cs: b and Cs-pure: c) due to the strong absorption in the range of 1630-1660cm-1,
which is characteristic for an imine group in CsSB (Guo, Xing, Liu, Ya, Wang, Li, et al., 2005).
All three spectra reveal the characteristic of the - RC=N- bonds, namely the absorption at
1641cm-1. The spectra of CsSBRTBr (Fig.5, a) and CsSB80Br (Fig.5, b) are identical with the
spectra in Fig. 4 (a, b); spectra of CsSBVinyl-1 (Fig.4, c) and CsSBVinyl-2 (Fig.5, c) are also
identical. Spectra in Figures 4 and 5 demonstrate new low-intensity bands with an absorption
maximum of 1574cm-1, and within the range of 720 - 850cm-1.
In CsSB molecule synthesized with salicylic aldehyde, or its derivatives as carbonyl
compounds (Guinesi & Cavalheiro, 2006; Santos, Dockal, & Cavalheiro, 2005), the absorption at
1540-1590cm-1 is attributed to the C=C stretching vibration in the aromatic ring of the aldehyde,
while the absorption at 700-800cm-1 is attributed to the out-of-plane bending vibrations of the
aromatic C–H bonds. The absorptions at 1602cm-1, 1514cm-1 and 1452cm-1, as well as within the
range of 700-850cm-1, are also present in the N-aryl spectra of Cs (Sajomsang, Tantayanon,
Tangpasuthadol, Thatte, & Daly, 2008). These authors attributed them to C=C and C-H bonds in
the aromatic rings and to the out-of-plane deformation vibrations of the aromatic C–H bonds. It
is noteworthy that Cn, Cs and Cs-pure obtained by us do not show any noticeable absorption in
the range of 720-850cm-1 (Fig.3. a, b and c, respectively). The comparison of the zoomed-in
FTIR spectra sections within 650-850cm-1 (Fig.4) revealed some spectral features of CsSBRTCl
(a), CsSB80Cl (b) and CsSBVinyl-1 (c). As it is shown in Figure 6, the absorption with a
maximum at 822cm-1 is split into two components: 822cm-1 and 804cm-1 in spectrum (b),
whereas the maximum of spectrum (c) is shifted to 804cm-1 and has a very small shoulder at
822cm-1. The absorption maxima at 704cm-1 and 739cm-1 are the same in all three spectra
(Fig.6). The same is true for the FTIR spectra shown in Figure 7. As compared with and as
distinct from Figure 6, Figure 7 reveals a small shoulder at 804cm-1 in spectra (a) and (b) that
transforms to the main absorption peak in spectrum (c) at approximately 800cm-1.
According to the literature data, in a graft polymer of Cs with the methacrylic acid (Pati &
Nayak, 2011), the vinyl group show an absorption at 809cm-1, and in Cn–methacrylate (Khor,
Wu, Lim, & Guo, 2011), the vinyl group shows an absorption maximum at 815cm-1. Comparing
our spectra with the published ones (Figs.6 and 7), we can assume that in our products
(CsSBVinyl-1 and CsSBVinyl-2) the absorption maximum at 804cm-1 corresponds to the vinyl
group. Apparently, change in temperature at which the interaction of Cs with aldehyde-1 and
aldehyde-2 takes place, from RT to 80oC, causes a partial dehydrohalogenation and formation of
the vinyl group in trace amounts. An evidence for this is the spectrum (b) in Figure 6. Most
likely, the absorption maximum at 822cm-1 corresponds to the C-C bending vibrations in the
halogenalkyl group; as it is shown (Reaction 2), dehydrohalogenation of CsSB in an alkaline
environment produces the vinyl groups. The spectra in Fig.6 and Fig.7 show the formation of the
vinyl groups in CsSBVinyl-1 and CsSBVinyl-2.
1H NMR spectral data also confirm the formation of products shown in Reactions 1 and 2
(Table 3). The correlation of signals with corresponding protons was determined on the basis of
the data provided in the previous reports (Lavertu et al., 2003; Santos, Dockal, & Cavalheiro,
1
2005; Soliman, El-Kousy, Abd-Elbary, & Abou-zeid, 2013). From H NMR spectra, DS of the
synthesized Schiff bases was determined by using a known equation (Santos, Dockal, &
Cavalheiro, 2005). It is calculated that DS for CsSBRTCl and CsSB880Cl is 8.3 and 9.6%,
respectively. For CsSBRTBr and CsSB80Br, DS values are equal to 8.8 and 10.4%, respectively.
It is interesting to note that the reaction temperature increase, from 20°C to 80°C, slightly
influences DS in CsSBRTCl and CsSB80Cl, as well as in CsSBRTBr and CsSB80Br. Minor

changes in DS with temperature were explained by the low activation energy of the amine
reaction with aldehydes as carbonyl compounds (Eldin, Hashem, Omer & Tamer, 2015).
X-ray diffractograms for CsSB products are presented in Figure 8. For Cn (Fig. 8, a), Cs
(Fig. 8, b) and Cs-pure (Fig. 8, c) samples, the reflection maxima were revealed at 2θ of
approximately 20º. These reflections are characteristic for Cn, Cs and idexed as 110 in (Zhang,
Xue, Xue, Gaoa & Zhang, 2005; Sashikala & Shafi, 2014); they practically disappear for CsSB
samples (Fig. 8, d, e, f, g, h and i), therefore, in general terms, samples (d) to (i) have amorphous
structure. Apparently, the presence of hydrophobic 41-(2-halogenethyl) phenyl and 41-
vinylphenyl substituents in CsSB macromolecules destroys the intra- and intermolecular
hydrogen bonding and, as a result, the crystal structure partially transforms into an amorphous
one.
To study antibacterial properties of the CsSB derivatives, same classic species of bacteria
were chosen, such as E. coli, Pseudomonas aeruginosa, as well as SP Pseudomonas. The
efficiency of extraction of select metal ions from various environments by means of synthesized
CsSB derivatives is being studied.
4. Conclusions
Cs is obtained by deacetylation of Cn extracted from the armors of crustaceans Astacus
leptodactylus (lake Sevan, Armenia). The interaction of the Cs with aromatic aldehydes produces
chitosan Schiff bases (CsSB) in a mild acidic environment. Having in chitosan-derived Schiff
bases the pending vinyl groups, which are readily available for polymerization and
copolymerization reactions, creates an opportunity for assembling novel polymeric structures
which in turn can exhibit properties of immediate interest to chemistry, biology, and medicine.
Acknowledgements
The work is performed within the basic funding program from AANL-YerPhI-foundation of
the Government of Armenia and the Armenian National Science & Education Fund (ANSEF;
award 2015, microbio – 3871). I would like to thank Gagik Melikyan, Aram Petrosyan, Vahram
Ghazaryan, Suren Kharatyan, Vardan Tadevosyan, Jirayr Levonyan, Valeri Bagdasaryan and
Gagik Papyan for their help at different stages of this study.

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Fig.1. TG, DTA, DTG curves of Cn (a), Cs (b) and Cs - pure (c)

Fig.2. TG, DTA, DTG curves of CsSBRTCl (a), CsSB80Cl (b) and CsSBVinyl-1(c).

Fig.3. FTIR spectra of Cn (a), Cs (b) and Cs – pure (c).
Fig.4. FTIR spectra of CsSBRTCl (a), CsSB80Cl (b) and CsSBVinyl-1(c).

Fig.5. FTIR spectra of CsSBRTBr, CsSB80Br (b) and CsSBVinyl-2 (c).

Fig.6. FTIR spectra of CsSBRTCl (a), CsSB80Cl (b) and CsSBVinyl-1 (c) zoomed in the range
of wavenumbers within 650 – 850cm-1.

Fig.7. FTIR spectra of CsSBRTBr, CsSB80Br (b) and CsSBVinyl-2 (c) zoomed in the range of
wavenumbers within 650 – 850cm-1.

Fig.8. X-ray diffractograms of Cn (a), Cs (b), Cs-pure (c), CsSBRTCl (d), CsSB80Cl (e),
CsSBVinyl-1 (f), CsSBRTBr (g), CsSB80Br (h), CsSBVinyl-2 (i).
Reaction 1.
OH
OH
OH
T°C
O
O
O
+
O
CH
(CH2)2
X
O
HO
O
HO
O
_
O
H2O
HO
NH2
NHCOMe
NH2
n
4-(2-halogenethyl) benzaldehyde
OH
OH
OH
5
4
6
O
O
O
O
HO
O
HO
O
O
HO
2
3
1
N
NHCOMe
N
n
CHR
7 CHR
Schiff's base
9
10
12
14
15
8
11
R =
CH2 CH2
X
13
X = Br; Cl.
T°C = 22 (RT); 80.
Reaction 1. Synthesis of chitosan Schiff bases (CsSB) by interaction of Cs with 4-
(2-halogenethyl) benzaldehydes (aldehyde-l and aldehyde-2).

Reaction 2.
OH
OH
OH
O
O
O
T°C, NaOH/H2O
O
HO
O
HO
O
O
_
H X
HO
N
NHCOMe
N
n
CHR
CHR
Schiff's base
OH
OH
OH
4
6
O
O
O
N
5
O
HO
O
HO
O
O
HO
2
3
1
N
NHCOMe
n
CH
7 CH
8
13
12
9
10
15
11
14
CH CH2
CH CH2
T°C = 10.
NaOH/H2O = 3.75g/19ml.
H X = HBr; HCl.
Reaction 2. Synthesis of Vinyl derivative (CsSBVinyl) from dehydrohalogenation
of chitosan Schiff bases (CsSB).

Table1. Elemental analysis data.
Sampl
e
obtained
calculated*
% O % Cl % Br C/N
Yield
%
% C
% N
% H
C/N
6.64
% C
% N
% H
6.48
Cn,
DA-
90.5%
Cs -
46.33
6.98
7.70
6.36
47.09 7.03
39.40
-
-
6.70
-
-
45.39
6.58
5.89
46.09 7.73
6.65
39.53
-
-
5.96
DA-
47.6%
CsSBR 47.80
T- Cl
CsSB8 46.43
0 - Cl
CsSB- 45.87
Vinyl-
7.57
7.34
7.43
6.69
6.50
6.46
6.18
6.31
6.17
46.98 7.46
47.12 7.42
47.25 7.54
6.58
6.57
6.62
38.15
37.94
38.60
0.82
0.94
-
-
-
-
6.30
6.35
6.26
9.4
7.1
64.8
1
CsSBR 45.72
T - Br
CsSB8 47.46
0 - Br
CsSB- 46.55
Vinyl-
7.35
7.18
7.50
6.50
6.35
6.58
6.23
6.61
6.20
46.53 7.36
46.61 7.30
47.18 7.55
6.51
6.48
6.62
37.66
37.34
38.65
-
-
-
1.94 6.32
2.27 6.38
6.4
8.3
-
6.25
89.8
2
*- in calculations the content of water isn't considered.

Table 2. Solubility of synthesized products.
Solvents
DMSO DMSO +
Sample
CF3COOH
CH3COOH
HCl
NaOH
KOH
H2O
CF3COOH
RT
RT
T,700 (Ice
(32.4%
÷0.1%)
RT
(1.5%÷
0.2 %)
RT
RT
(0.3%). (0.3%). RT
RT RT
C
)
RT
CsSBRT part.s part.s In-
part.sol.
and s.
part.sol part.sol
soluble
part.sol part.sol part.s
ol.
Cl
ol
ol.
le
.
. ***
.
.
and
s*
and s. **
and s.
CsSB80
Cl
part.s part.s In-
ol ol. le
and s and s.
part.sol.
and s.
part.sol part.sol
and s.
soluble
soluble
part.sol part.sol part.s
.
.
.
ol.
CsSB-
Vinyl-1
part.s part.s in-
ol ol. le
and s and s.
part.sol.
and s.
part.sol part.sol
.
.
In-le
In-le
In-le
and s.
CsSBRT part.s part.s In-
part.sol.
and s.
part.sol part.sol
soluble
soluble
part.sol part.sol part.s
ol.
Br
ol
ol.
le
.
.
.
.
and
and s.
and s.
CsSB80
Br
part.s part.s In-
ol ol. le
and s and s.
part.sol.
and s.
part.sol part.sol
part.sol part.sol part.s
.
.
.
.
ol.
and s.
CsSB-
Vinyl -2
part.s part.s inle part.sol.
ol ol. and s.
and s and s.
part.sol part.sol
soluble
In-le
In-le
In-le
.
.
and s.
part.sol and s* - partialy soluble and swell; in-le** - insoluble. part.sol.*** - partialy soluble.

Table 3. 1H NMR data of synthesized products.
Chemical shift δ, (ppm)
Sample,
solvent
H7
H9,H10,
H12,H13
H14
H15a
H15b
H1
5H,
H3-2H6
H2
2H,
H14
3H,
COMe
-
-
-
-
-
-
4.75
3.2÷3.8
3.2÷3.8
3.2÷3.8
2.89
1.83
1.80
1.90
Cs,
DMSO-d6
/CF3COOD
Cs –pure,
D2O/CF3CO
OD
-
-
-
-
-
-
4.56
4.60
2.87
2.87
9.27
7.10÷7.20
and
6.60÷6.70
-
-
-
-
-
-
2.40
CsSBRTCl,
D2O
9.27
9.27
7.10÷7.20
and
6.65÷6.70
7.10÷7.20
and
4.60
4.60
3.2÷3.8
3.2÷3.8
2.89
2.90
2.40
-
1.90
1.95
CsSB80Cl,
D2O
6.60
5.70
5.20
CsSBVinyl-
1,
D2O/CF3CO
OD
CsSBRTBr,
D2O/CF3CO
OD
CsSB80Br,
D2O/CF3CO
OD
6.65÷6.75
-
-
-
-
-
-
9.40
9.40
9.40
7.70÷7.90
and
7.30÷7.40
4.70
4.70
4.60
3.2÷3.8
3.2÷3.8
3.2÷3.8
. 2.85 2.50
1.93
1.95
1.95
7.70÷7.90
and
7.30÷7.40
2.85
2.90
2.50
-
7.10÷7.20
and
6.65÷6.75
6.60
5.70
5.20
CsSBVinyl-
2, DMSO-
d6
/CF3COOD