Synthesis and characterization of N-propyl-N-methylene phosphonic chitosan derivative

Article abstract of DOI:10.1016/j.carbpol.2009.08.011

A simple methodology for the preparation of a new chitosan derivative called N-propyl-N-methylene phosphonic chitosan (PNMPC) is proposed. Introduction of a propyl chain onto a modified chitosan (N-methylene phosphonic chitosan) offers the presence of hydrophobic and hydrophilic branches for controlling solubility properties of the new derivative. Its chemical identity was determined by FT-IR, ¹H, ¹³C and ³¹P NMR spectroscopy. The degree of propyl substitution estimated by elemental analysis was 0.64. Furthermore derivative molecular weight is about 60 × 10³, X-ray diffraction and SEM showed certain degree of crystallinity and homogeneous surface with a rather packed structure. This derivative opens new perspectives in food, pharmaceutical and cosmetic fields.

Full text of DOI:10.1016/j.carbpol.2009.08.011

Carbohydrate Polymers 79 (2010) 475–480
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Short communication
Synthesis and characterization of N-propyl-N-methylene phosphonic
chitosan derivative
*
Adriana Zuñiga , Adriana Debbaudt, Liliana Albertengo, María Susana Rodríguez
Instituto de Química (INQUISUR), Universidad Nacional del Sur-CONICET, Av. Alem 1253, (8000) Bahía Blanca, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple methodology for the preparation of a new chitosan derivative called N-propyl-N-methylene
phosphonic chitosan (PNMPC) is proposed. Introduction of a propyl chain onto a modified chitosan
(N-methylene phosphonic chitosan) offers the presence of hydrophobic and hydrophilic branches for con-
Received 15 December 2008
Received in revised form 30 July 2009
Accepted 6 August 2009
trolling solubility properties of the new derivative. Its chemical identity was determined by FT-IR, 1H, 13
C
Available online 12 August 2009
and 31P NMR spectroscopy. The degree of propyl substitution estimated by elemental analysis was 0.64.
Furthermore derivative molecular weight is about 60 ꢀ 103, X-ray diffraction and SEM showed certain
degree of crystallinity and homogeneous surface with a rather packed structure. This derivative opens
new perspectives in food, pharmaceutical and cosmetic fields.
Keywords:
Chitosan
Derivative
Solubility
Ó 2009 Elsevier Ltd. All rights reserved.
Amphiphilic properties
N-Methylene phosphonic chitosan
1. Introduction
the hydrophobic moiety counterbalances the electrostatic repul-
sion but it also gets more tensioactive properties.
Chitosan, the deacetylated derivative of chitin, is a natural bio-
polymer consisting of b-1-4 linked N-acetyl glucosamine (GlcNAc)
and glucosamine (GlcN) repeating units. It has a high molecular
weight resulting in its low solubility in most solvents and shows
bioactivity only in acidic medium, these reasons limit its applica-
tions especially in medicine and food industry.
In this paper, we report the successful preparation of an N-alkyl
derivative of the water soluble NMPC using a reductive N-alkyl-
ation with propyl aldehyde to obtain a new amphiphilic hybrid
material of synthetic and natural polymer so-called N-propyl-N-
methylene phosphonic chitosan (PNMPC).
To increase aqueous solubility and to improve biological, chem-
ical and physical properties, many derivatives of chitosan have
been synthesized (Alves & Mano, 2008; An, Dung, Thien, Dong, &
Nhi, 2008; Dung, Milas, Rinaudo, & Desbriéres, 1994; Ma et al.,
2008; Mourya & Inamdar, 2008; Rinaudo, 2006; Sui, Wang, Dong,
& Chen, 2008).
In a previous work we described the synthetic strategy of a no-
vel soluble chitosan derivative: the N-methylene phosphonic
chitosan (NMPC) by the transformation through an additional
functional group in a homogeneous, one step, reaction system for
the purpose of creating a chitosan derivative that allowed solubil-
ity in water under neutral conditions (Heras, Rodríguez, Ramos, &
Agulló, 2001). Lately a methodology was developed for the prepa-
ration of a derivative carrying alkyl and phosphonic groups (LMPC).
On the LMPC the addition of alkyl groups seems to weaken the
hydrogen bond and provides good solubility in organic solvents
(Ramos, Rodríguez, Rodríguez, Heras, & Agulló, 2003). Moreover,
the new derivative proves to be an amphiphilic system in which
2. Materials
2.1. Preparation of chitin and chitosan
Chitin was isolated from shrimp shells waste (Pleoticus mülleri).
It was homogenized and rinsed with water to remove the organic
material, then treated with 9% (w/w) NaOH at 65 °C for 90 min, to
remove proteins and demineralized with 10% (v/v) HCl at 20 °C for
15 min, washed until neutral pH and dried.
Chitosan was prepared by heterogeneous deacetylation of chitin
at 136 °C with 50% (w/w) NaOH for 1 h.
2.2. Synthesis of N-methylene phosphonic chitosan (NMPC)
A solution of phosphorous acid/water (1:1 w/w) was added
dropwise with stirring, at room temperature for 1 h to chitosan
2% (w/v) in glacial acetic acid 1% (v/v). The temperature of the reac-
tion vessel was raised to 70 °C and one part of formaldehyde 36.5%
(by weight) was added dropwise over 1 h with reflux and left over-
night at the same temperature. Solution was dialyzed against
* Corresponding author. Tel.: +54 291 4595100; fax: +54 291 4595187.
E-mail address: azuniga@criba.edu.ar (A. Zuñiga).
0144-8617/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2009.08.011

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A. Zuñiga et al. / Carbohydrate Polymers 79 (2010) 475–480
demineralized water for 48 h or until pH was raised to 6.8 in dial-
ysis tubing with a cut off value of 12,400 Da. Finally solution was
freeze-dried (Heras et al., 2001).
OH
H
O
2.3. Synthesis of N-propyl-N-methylene phosphonate chitosan
(PNMPC)
O
HO
NR1R2
n
NMPC (1 g) was suspended in 100 mL distilled water–methanol
(1:1 v/v), propyl aldehyde (1.5 g) was added and stirred for 30 min.
Reduction was carried out with an excess of sodium borhydride
which was added for 2 h and then left stirring overnight at room
temperature. PNMPC sodium salt was obtained by dialyzing the
reaction mixture against demineralized water for 48 h or until a
water pH of 6.8 (dialysis tubing with a MW cut off value of
12,400). The solution was freeze-dried.
[A-/-B-/-C-/-D-/-E-/F…]n
R1
R2
A
B
C
D
E
F
-H
-H
-COCH3
-H
-H
-CH2-PO3H2
-CH2-PO3H2
-CH2-CH2-CH3
-CH2-CH2-CH3
-CH2-PO3H2
-H
-CH2-CH2-CH3
Fig. 1. Chemical structure of N-propyl-N-methylene phosphonic chitosan.
3. Methods
3.1.4. Solubility test
Solubility of PNMPC in different solvents was evaluated. Solu-
tions of 10 mg of the polymer in 5 mL of each solvent were prepared.
3.1. Characterization of PNMPC
3.1.1. X-ray diffraction spectrometry
X-ray diffraction spectrometry data were collected using a Rig-
aku D-Máx. III C diffractometer (Cu Ka) irradiated at 35 kv–15 ma.
3.1.5. Molecular weight determination
Weight-average molecular weight (Mw), number-average
molecular weight (Mn) and molecular weight dispersion (Mw/Mn)
were determined by a Waters-Breeze gel permeation chromato-
graph (Model 1525), connected to a Waters 2414 (Mod.410) Differ-
ential refractometer and a Dawn DSP Light scattering detector. A
set of five columns connected in series Ultrahydrogel, Waters
7.8 ꢀ 300 mm of 120, 250, 500, 1000 and 2000 Å size pore were
used. The temperature was maintained at 30 °C. The eluent was
CH3COOH/CH3COONa pH 4.8, standards were Pullulans (Shodex
Standard P-82. No. 30901-Showa Denko).
3.1.2. NMR spectroscopy
13C, 1H and 31P NMR spectra were recorded on a Varian VNMRS-
400 instrument spectrometer at 70 °C. PNMPC (23 mg) was dis-
solved in 0.5 ml of 5% (w/w) DCl/D2O at 70 °C. Chemical shift val-
ues were recorded downfield from trimethylsilyl propionate
sodium salt (TSP) as standard and PO4H3 (85%) for the 31P NMR
spectrum. The heterocorrelation was done with a Bruker DMX-
500 instrument.
3.1.3. IR spectroscopy
3.1.6. Elemental analysis
The spectrum was recorded on a Nicolet FT-IR instrument. The
KBr discs were prepared by blending anhydrous KBr with PNMPC
(1%).
Elemental analysis were carried out with a Carlo Erba 1108
instrument. Gas separation was done by a gas chromatograph with
a variable length Porapak column and a TCD detector.
Fig. 2. FT-IR spectrum of PNMPC.

A. Zuñiga et al. / Carbohydrate Polymers 79 (2010) 475–480
477
Table 1
13C NMR chemical shifts of PNMPC.
Carbon
C1 (C)
C1 (F)
99.49
C1 (D + E)
98.00
C2
C3
C4
C5
C6
CH2 (D)
51.70
CH2 (E)
45.24
d (ppm)
100.37
70.81
71.49
71.07
72.52
79.69
80.90
77.64
63.89
Signals at 45.24, 22.24 and 13.17 correspond to –CH3 and –CH2 of the propyl group.
200
150
100
500
0
102.0
ppm (t1)
101.0
100.0
99.0
98.0
97.0
96.0
95.0
94.0
Fig. 3. 13C NMR PNMPC spectrum expansion.
Fig. 4. 1H–13C NMR spectra.

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A. Zuñiga et al. / Carbohydrate Polymers 79 (2010) 475–480
3.1.7. Scanning electron microscopy
Scanning electron microscopy were carried out with a Scanning
electron microscope LEO mod. EVO 40 XVP.
nomethylphosphonic acids are greatly dependent upon the acidity
of the solution in which they are measured, such effect causes
downfield shifts (Moedritzer & Irani, 1966). In this study the poly-
mer was dissolved in D2O/DCl and shows a signal centered at
ꢁ0.004 ppm Assignments of 1H and 13C NMR spectra were con-
firmed by two-dimensional heteronuclear shift correlation spec-
troscopy (Fig. 4).
4. Results and discussion
The introduction of C3 chain onto NMPC’s free amino groups by
a reductive amine reaction leads to N-propyl-N-methylene phos-
phonic chitosan (PNMPC). The incorporation of N-methylene phos-
phonic group afforded the hydrophilic moieties and alkyl group the
hydrophobic one.
The substitution degree (SD) of the PNMPC was calculated by
comparing the C and N molar ratio obtained from the elemental
analysis in different derivatives. It can be observed an increase in
the molar ratio as a consequence of the introduction of the propyl
group. The degree of phosphonomethylation and alkylation were
stipulated to be 1.56 and 0.64, respectively (Table 2).
Fig. 1 shows its chemical structure with the possible combina-
tions of R1 and R2 substituents.
X-ray diffraction analysis (Fig. 5) showed that PNMPC white
powder has a certain degree of crystallinity. Two reflections fall
The FT-IR spectrum (Fig. 2) shows the axial stretching of OH
group in 3429 cmꢁ1 which appears superimposed to the NH
stretching band, an amide band (amide I) at 1568 cmꢁ1 and an ax-
ial deformation band due to C–N at 1409 cmꢁ1
.
The alkyl chain shows signals at 2966, 2924 and 2869 cmꢁ1 cor-
responding to CH3 and CH2 groups, besides the characteristic
deformation signals at 1460 and 1386 cmꢁ1
.
The carbonyl group due to NH–COCH3 group appears in
1650 cmꢁ1
.
This derivative also presents two phosphonic group characteris-
tics bands, one at 2353 cmꢁ1 (splitted) due to P–H stretching and
the other at 1067 cmꢁ1 corresponding to
m P–OH.
The assignments and chemical shift of 1H NMR signals are:
d = 5.09 (C and F, H1), 5.02 (E, H1); d = 4.31 (CH–N@C imine);
d = 4.06–3.80 (H3, H4, H5, H6), d = 3.38–3.28 (H2); d = 3.27 [N–
CH2–P, (D)] d = 3.07 [CH3–CH2–CH2–N, N–CH2–P (C)]; d = 2.06
(–NHCOCH3); d = 1.75 (CH3–CH2–CH2–N); d = 0.99 (CH3–CH2–
CH2–N).
On the 1H and 13C NMR spectra the replacement of the free ami-
no group of chitosan to obtain the NMPC derivative showed two
forms distinguishable: one was assigned to the monophosphonic
secondary amine and the other one to the tertiary diphosphonic
amine, showing that the time of reaction enhances the degree of
phosphonomethylation (Heras et al., 2001).
The signal at 5.09 ppm is the evidence of dialkylation of the
amine group which is rather increased due to the presence of a
minor percentage of the N-methylene phosphonic moiety. Desbriè-
res, Martínez, and Rinaudo (1999) noted an identical chemical shift
for a similar propyl derivative.
The signal at 5.02 ppm, of higher intensity, is assigned entirely
to the monopropyl form showing that it is the predominant group.
At 4.54 ppm there is a little signal corresponding to the N-ace-
tylglucosamine unit.
Table 1 shows the signals assignment of the 13C NMR spectrum.
The chemical shift at 99.49 ppm is due to the introduction of one
alkyl group. Signals at 45.24, 22.24 and 13.17 correspond to –CH3
and –CH2 of the propyl group.
An expansion of the spectrum allowed us to find that in
98.00 ppm there are evidences of a splitting which is the result
of the overlapping of the dialkylated moiety and the tertiary
diphosphonic amine. Furthermore there is a small signal due to
the monophosphonic secondary amine at 100.37 ppm (Fig. 3).
To confirm the presence of the phosphonic group a 31P NMR
spectrum was registered. The 31P NMR chemical shifts of
a-ami-
Table 2
Elemental analysis and substitution degree (SD) of chitosan and its derivatives.
C
N
C/N*
DS
Chitosan
NMPC
PNMPC
40.87
34.68
43.63
7.61
5.25
5.23
6.30
7.86
9.78
1.56
0.64
*
Molar ratio C/N.
Fig. 5. X-ray diffraction pattern of Chitosan, NMPC and PNMPC.

A. Zuñiga et al. / Carbohydrate Polymers 79 (2010) 475–480
479
at 2h: 7°720 and 20°030 for the alkylated derivative with an evident
increase of intensity of the second band, whereas chitosan and
NMPC only showed one broad peak at 2h: 21°44 and 22°720 respec-
tively. It may be concluded that the rise of crystallinity is a conse-
quence of the introduction of the short alkyl chain.
The phosphonomethylation reaction proved to decrease the
molecular weight of the original chitosan. This effect was more sig-
nificant with longer reaction times (Ramos et al., 2003). Consider-
ing the experimental conditions we expected a molecular weight
in the range of 50 ꢀ 103 Da. Mn, Mw and D (Mw/Mn) were deter-
mined by GPC. Figs. 6 and 7 show the signals obtained for PNMPC
determined with a differential refractometer and a light scattering
detector. Reproducibility of results obtained with both detectors at
different concentrations proves to be acceptable, indicating that
the main macromolecular structure of the derivative is about Mw
60 ꢀ 103 Da.
It is well known the effect of introducing hydrophobic branches
on chitosan derivatives for controlling the solubility properties.
Acetamido and primary groups of the polymer have an important
role in the formation of intra- and/or intermolecular hydrogen
bonding. The removal of the two hydrogens atoms of amino group
of chitosan and the introduction of some hydrophobic nature
group by chemical modification causes destruction of its crystal-
line structure resulting in the improvement of solubility in organic
solvents.
Table 3 shows PNMPC solubility at different temperatures: at
60 °C it proves to be soluble in HCl solutions (0.1 M and 1%) and
in organic solvents like acetone, ethanol, pyridine, DMF and DMSO.
Fig. 6. GPC/SEC-LS and DR signals for PNMPC.
PNMPC
Mn
Mw
D
Mass
[mg]
M P
Volumen
[mL]
Concentration
[mg/mL]
Samples
QNAc-C1
25,900
27,900
57,800
63,300
2.23
2.27
5.6
5.00
5.00
1.12
2.26
QNAc-C2
Inyection 1
11.3
QNAc-C2
Inyection 2
26,600
63,400
2.38
11.3
5.00
2.26
Fig. 7. GPC/SEC-DR compared chromatograms.
Table 3
Solubility test of PNMPC derivative.
NaOH 0.1 M
NaOH 1%
HCl 0.1 M
HCl 1%
Acetic acid 1%
DMF
DMSO
Acetone
Ethanol
Water
Pyridin
18 °C
40 °C
60 °C
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
+
+
++
++
++
++
++
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
+
ꢁ
ꢁ
+
+
+
+
ꢁ
++
Sample (10 mg) and 5 ml of solvent; ++, soluble; +, partially soluble; +ꢁ, swelled; ꢁ, insoluble.

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A. Zuñiga et al. / Carbohydrate Polymers 79 (2010) 475–480
Fig. 8. SEM images of PNMPC derivative.
PNMPC showed a rather similar behaviour to LMPC derivative, syn-
thesized previously by our group, when its solubility was evalu-
ated with the same solvents (Ramos et al., 2003).
The SEM image presents a homogeneous surface with a rather
packed structure (Fig. 8).
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Financial support from Universidad Nacional del Sur is grate-
fully acknowledged.