January/February 2002 Published by the American Chemical Society Volume 3, Number 1

© Copyright 2002 by the American Chemical Society

Communications

Chemoselective Protection of the Amino Groups of Chitosan by

Controlled Phthaloylation: Facile Preparation of a Precursor
Useful for Chemical Modifications
Keisuke Kurita,*,† Hiroyuki Ikeda,† Yuya Yoshida,† Manabu Shimojoh,‡ and Manabu Harata†
Department of Applied Chemistry, Faculty of Engineering, Seikei University,
Musashino-shi, Tokyo 180-8633, Japan, and Research and Development Department, Toyo Suisan
Kaisha, Ltd., Kohnan, Minato-ku, Tokyo 108-8501, Japan
Received July 20, 2001; Revised Manuscript Received September 26, 2001

A simple and convenient procedure for chemoselectively protecting the amino groups of chitosan has been
developed to provide N-phthaloyl-chitosan that is indispensable as a soluble N-protected precursor for further
controlled modification reactions of chitosan. Although the conventional N-phthaloylation of chitosan in
N,N-dimethylformamide was accompanied by partial phthaloylation of the hydroxy groups, the addition of
a small amount of hydroxy-containing compounds effectively suppressed the O-phthaloylation. Of some
compounds examined, water proved particularly suitable, resulting in the formation of chemoselectively
N-phthaloylated chitosan without any appreciable O-phthaloyl groups. The resulting N-phthaloyl-chitosan
was found to be crystalline despite the presence of a bulky substituent. A solubility test indicated that
N-phthaloyl-chitosan exhibited considerable affinity for organic solvents.

Introduction chitin,4 tosyl-chitin,5 iodo-chitin,5 and trimethylsilyl-chitin.6

Despite unique biological activities and physicochemical
properties,1 chitin still remains an unutilized biomass resource
due to its intractable nature; it is insoluble in common
solvents. In view of developing materials with advanced
functions, many attempts have been made to modify the
molecular structure of chitin and thereby to improve or
control the properties.2 The reactions, however, often en-
counter difficulty because of the heterogeneous reaction
conditions, multifunctionality with three kinds of functional
groups, and poor reactivity. The products are therefore
structurally ambiguous in many cases, and it is difficult to
discuss the structure-property relationship.
Some soluble derivatives of chitin have proved useful for
performing modification reactions in a facile and controlled
manner.3 They include partially deacetylated water-soluble
* To whom all correspondence should be addressed.
† Seikei University.
‡ Toyo Suisan Kaisha.
10.1021/bm0101163 CCC: $22.00
Published on Web 11/21/2001
Phthaloylated chitosan is a particularly important and indis-
pensable organosoluble precursor.7 Treatment of chitosan
with phthalic anhydride, however, generally results in partial
O-phthaloylation in addition to the N-substitution.8 With the
phthaloylated product as a key intermediate, various modi-
fication reactions proceed smoothly in solution,9-11 but the
O-phthaloyl group is an obstacle in most cases to quantitative
and regioselective substitution. Recently we reported on the
removal of O-phthaloyl groups by transesterification,12 but
it is desirable to avoid the use of alkali that might be harmful
to the N-phthaloyl group. If N-phthaloyl-chitosan can be
prepared in a one-step reaction, it would be highly beneficial
for conducting a wide variety of modification reactions site-
specifically and quantitatively to construct well-defined
molecular architectures on chitosan. We report here a simple
and reliable method to provide chemoselectively protected
N-phthaloyl-chitosan and discuss some properties of the
product.
© 2002 American Chemical Society
2 Biomacromolecules, Vol. 3, No. 1, 2002 Communications

Table 1. Phthaloylation of Chitosan in Various Solventsa

solvent (v/v) time (h) dsb
DMF 5 1.54c
DMF 8 1.30d
DMF 24 1.16d
DMF/ethanol (95/5) 8 0.65e
DMF/ethylene glycol (95/5) 8 0.50f
DMF/Methyl Cellosolve (95/5) 8 0.80g
DMF/water (95/5) 8 1.00h
a The reaction was carried out with 0.300 g of chitosan and 3 equiv of

phthalic anhydride in 6 mL of solvent at 120 °C. b Degree of substitution

calculated from the C/N value of elemental analysis. c Calcd for
(C14H13NO6)0.46(C22H17NO9)0.54‚1.7H2O: C, 56.11; H, 4.49; N, 3.57. Found:
C, 56.09; H, 4.33; N, 3.57. d Reference 12. e Calcd for (C6H11NO4)0.35-
(C14H13NO6)0.65‚0.9H2O: C, 51.36; H, 5.43; N, 5.35. Found: C, 51.38; H,
5.56; N, 5.32. f Calcd for (C6H11NO4)0.50(C14H13NO6)0.50‚H2O: C, 49.18;
H, 5.78; N, 5.73. Found: C, 49.21; H, 5.97; N, 5.75. g Calcd for
(C6H11NO4)0.20(C14H13NO6)0.80‚0.8H2O: C, 53.26; H, 5.12; N, 5.01. Found:
C, 53.26; H, 5.13; N, 5.02. h Calcd for C14H13NO6‚0.7H2O: C, 55.34; H,
4.78; N, 4.61. Found: C, 55.31; H, 4.64; N, 4.64.

Experimental Section

General Information. IR spectra were recorded on a

Shimadzu FTIR-8900 by the KBr method. 13C cross polar-
ization magic angle spinning (CP/MAS) NMR spectra were
taken with a JEOL JNM-LA400D at 20 °C and 13C frequency
of 100.40 MHz with TOSS (total suppression of sidebands)
and TOSDL (TOSS and dipolar-dephasing) modes using
hexamethylbenzene (17.36 ppm) as the external standard.
Elemental analysis was carried out with a Perkin-Elmer 2400.
X-ray diffraction diagrams were obtained by the powder
method with the use of Ni-filtered Cu KR radiation with a
MAC Science M03X-HF 1013 instrument. Phthalic anhy-
dride was of reagent grade and used as received. Solvents
were purified in usual manners and stored over molecular
sieves.
Phthaloylation of Chitosan. To a solution of 0.83 g (5.6
mmol) of phthalic anhydride in 6 mL of N,N-dimethylfor-
mamide (DMF) containing 5% (v/v) water was added 0.300
g (1.86 mmol pyranose) of fully deacetylated chitosan,10 and
the mixture was heated in nitrogen at 120 °C with stirring.
After 8 h of reaction, the resulting pale tan mixture was
cooled to room temperature and poured into ice water. The
precipitate was collected on a filter, washed with 150 mL
of methanol at room temperature for 1 h, and dried to give
0.444 g of the product as a pale tan powdery material. The
degree of substitution (ds) was determined to be 1.00 from
the C/N value of elemental analysis (see Table 1), and the
yield was 81.1% on the basis of the ds value. IR (KBr): ν
3450 (OH), 1776 (imide CdO), 1712 (imide CdO), 1150-
1000 (pyranose), and 721 cm-1 (arom). 13C CP/MAS
NMR: (TOSS mode) δ 57.29 (C-2), 61.76 (C-6), 72.44 (C-
3), 76.04 (C-5), 83.91 (C-4), 102.27 (C-1), 123.93, 131.80,
and 134.42 (Phth phenylene), and 169.01 ppm (Phth CdO);
(TOSDL mode) δ 131.70 (Phth C-1,2) and 169.10 ppm (Phth
CdO).
Results and Discussion
Protection of the functional groups of chitosan is crucial
for conducting modification reactions in a well-controlled
manner, and introduction of the phthaloyl group at the amine
functionality is ideal not only for the protection but also for
Figure 1. IR spectra of phthaloylated chitosans prepared by the
reaction at 120 °C (A) for 5 h in DMF, (B) for 8 h in DMF/ethanol
(95:5), and (C) for 8 h in DMF/water (95:5).
improving solubility.7 Furthermore, dephthaloylation is facile
with hydrazine at 80 °C.7,10 Although the phthaloylation is
accompanied by partial O-phthaloylation, the product is still
a convenient organosoluble precursor for some modification
reactions including acylation to prepare derivatives having
liquid crystalline properties.8,13 However, to expand the scope
of chemoselective and quantitative modification reactions of
chitosan, it would be highly advantageous to use structurally
well-defined N-phthaloyl-chitosan.
Phthaloylation Reaction. Phthaloylation of chitosan is
usually performed in DMF with excess phthalic anhydride
at 120-130 °C for 8 h.7 As shown in Table 1, the ds of the
product obtained after 5 h reaction was 1.54. Interestingly,
however, the ds decreased to some extent on prolonged
reaction. This implies the possibility of hydrolytic cleavage
of the once formed ester linkages with the water produced
by N-phthaloylation.
The reaction was thus examined in a mixed solvent
composed of DMF and a hydroxy-containing substance for
the possibility of chemospecific protection, and the results
are included in Table 1. The ds values of the products
prepared in DMF/ethanol and DMF/ethylene glycol were
low. Although the product prepared in DMF showed weak
bands at 2600-2700 cm-1 (free carboxyl) and medium ones
at 1290 and 1260 cm-1 (ester) in the IR spectrum in Figure
1, the products obtained in these mixed solvents showed
weak bands at 1290 and 1260 cm-1. This suggests incomplete
cyclization at the amino group and/or partial O-phthaloylation
though to only a small extent. Methyl Cellosolve appeared
to be better as a cosolvent, judging from the IR spectrum of
the product, but the ds was still less than 1.0.
Of the four cosolvents examined, water proved the most
appropriate; the product was much lighter in color, and
Communications
Scheme 1
furthermore, the ds of the product was confirmed to be 1.0
(Scheme 1). These data indicate that DMF/water is by far
the most suitable for phthaloylation in terms of the ds value
as well as the reaction selectivity, and the chemoselective
full N-phthaloylation became possible in a simple manner.
The yield was in a range 80-90%.
The IR spectrum (Figure 1C) of the product obtained in
DMF/water shows bands assignable to N-phthaloyl-chitosan
and no appreciable bands due to free carboxyl groups. The
twin absorptions at 1776 and 1712 cm-1 are characteristic
of imide; it should be noted that the latter is particularly sharp
in comparison with that in spectrum A, supporting the
absence of the ester carbonyl.
The solid-state 13C NMR spectra of the product are shown
in Figure 2. The spectrum in the TOSS mode exhibits peaks
assignable to N-phthaloyl-chitosan. In the TOSDL mode,
peaks due to CH and CH2 should disappear because of the
short relaxation times, and as expected, only two peaks
Figure 2. 13C CP/MAS NMR spectra of N-phthaloyl-chitosan pre-
pared by the reaction at 120 °C for 8 h in DMF/water (95:5) in (A)
TOSS mode and (B) TOSDL mode.
Biomacromolecules, Vol. 3, No. 1, 2002 3
ascribable to the carbonyl and C-1,2 of phthaloyl could be
observed. Moreover, the peaks in the spectra of both modes
were rather sharp and well resolved compared to those of
the products having additional O-phthaloyl groups.
Some Properties. As shown in Figure 3, fully deacety-
lated chitosan prepared by repeated alkali treatments showed
lower crystallinity than the ordinary chitosan of around 95%
deacetylation.14 The phthaloylated product prepared in DMF
was amorphous, because of the heterogeneous structure
owing to partial O-substitution in addition to the bulkiness
of the phthaloyl group. In sharp contrast, N-phthaloyl-
chitosan prepared in DMF/water showed certain crystallinity
as evidenced by diagram C in Figure 3, despite the
introduction of the phthaloyl group; this also supports the
uniform structure of the product. It is noteworthy that
N-phthaloyl-chitosan exhibited crystallinity even though such
a bulky substituent had been introduced.
A qualitative solubility test indicated that the resulting
N-phthaloyl-chitosan was soluble in some organic solvents
Figure 3. X-ray diffraction diagrams of (A) fully deacetylated chitosan,
(B) phthaloyl-chitosan (ds 1.54) prepared in DMF, and (C) N-phthaloyl-
chitosan (ds 1.00) prepared in DMF/water (95/5).
4 Biomacromolecules, Vol. 3, No. 1, 2002
including m-cresol, dichloroacetic acid, N,N-dimethylaceta-
mide/8% LiCl,15 and methanol/CaCl2.16 It swelled in more
common solvents such as pyridine, DMF, and dimethyl
sulfoxide that dissolved the product having additional O-
phthaloyl groups. The relatively low solubility of N-phtha-
loyl-chitosan may be partly attributable to some crystallinity
as suggested by X-ray diffractiometry.
Conclusions
Chemoselective N-phthaloylation of chitosan could be
accomplished successfully in one step using DMF containing
5% water as a solvent. It is rather surprising that crystallinity
was observed with N-phthaloyl-chitosan. Crystallization of
chitin or chitosan derivatives, particularly those having bulky
substituents, is usually difficult, and N-phthaloyl-chitosan
appears to be the first example of a derivative with a bulky
substituent that can crystallize. The N-phthaloyl-chitosan
exhibited high affinity for organic solvents, although some-
what lower than that of the product having additional
O-phthaloyl groups. The simple procedure established here
enables facile preparation of N-phthaloyl-chitosan, a conve-
nient precursor for the construction of sophisticated molecular
architectures based on the specialty biopolymer chitosan.
Acknowledgment. This work was supported in part by
a Grant-in-Aid for Scientific Research (No. 12650872) from
the Ministry of Education, Science, and Culture of Japan
and by a grant from Towa Shokuhin Kenkyu Shinkoukai.
References and Notes
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