New cationic hydrophilic and amphiphilic polysaccharides synthesized by one pot procedure

Article abstract of DOI:10.1016/j.carbpol.2010.06.027

Synthesis of cationic polysaccharides carrying quaternary ammonium groups of various chemical structures was performed by one pot procedure involving the chemical modification of a neutral polysaccharide (dextran, pullulan) with an equimolar mixture epichlorohydrin/tertiary amine, in aqueous media. Study of the reaction mechanism showed that the quaternization reagent was the glycidyl derivative of the tertiary amine formed in situ. Formation of cationic polysaccharides and their chemical structure were confirmed by elemental analysis, potentiometric titration, FT-IR and NMR spectroscopy. The reaction efficiency of the one pot procedure was similar to other methods using preformed clorohydroxypropyl or glycidyl quaternary ammonium reagents, but gave better results for the introduction of quaternary ammonium groups with more hydrophobic substituents. The procedure allowed the synthesis of a large variety of hydrophilic and amphiphilic cationic polysaccharides with potential application as hipolipemic drugs, flocculants or antibacterial agents.

Full text of DOI:10.1016/j.carbpol.2010.06.027

Carbohydrate Polymers 82 (2010) 965–975
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
New cationic hydrophilic and amphiphilic polysaccharides synthesized
by one pot procedure
Marieta Nichifor∗, Magdalena Cristina Stanciu, Bogdan C. Simionescu
“Petru Poni” Institute of Macromolecular Chemistry, Aleea Grigore Ghica Voda 41 A, 700484 Iasi, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of cationic polysaccharides carrying quaternary ammonium groups of various chemical
structures was performed by one pot procedure involving the chemical modification of a neutral polysac-
charide (dextran, pullulan) with an equimolar mixture epichlorohydrin/tertiary amine, in aqueous media.
Study of the reaction mechanism showed that the quaternization reagent was the glycidyl derivative of
the tertiary amine formed in situ. Formation of cationic polysaccharides and their chemical structure were
confirmed by elemental analysis, potentiometric titration, FT-IR and NMR spectroscopy. The reaction
efficiency of the one pot procedure was similar to other methods using preformed clorohydroxypropyl
or glycidyl quaternary ammonium reagents, but gave better results for the introduction of quaternary
ammonium groups with more hydrophobic substituents. The procedure allowed the synthesis of a large
variety of hydrophilic and amphiphilic cationic polysaccharides with potential application as hipolipemic
drugs, flocculants or antibacterial agents.
Received 30 March 2010
Received in revised form 20 May 2010
Accepted 14 June 2010
Available online 19 June 2010
Keywords:
Cationic polysaccharide
Chemical modification
Dextran
Pullulan
Self-assembling polymers
Anticholesteremic
Antibacterial agents
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
available reagents such as N-(3-chloro-2-hydroxypropyl)-
N,N,N-trimethylammonium chloride (CHPTAC) ((QUAB®188))
Polysaccharides containing quaternary ammonium groups are
hydrophilic, biodegradable, bacteriostatic, and can support some
components of the skin and the hair (Brode, Goddard, Harris, &
Salensky, 1991). These polymers have numerous applications in a
variety of fields, including the paper and textile, food, cosmetics,
chemical, and pharmaceutical industries (Belalia, Grelier, Benaissa,
& Coma, 2008). Due to their ammonium groups, cationic polysac-
charides can control the surface charge of particles in aqueous
suspensions, and have a great potential in clarification of raw and
industrial wastewater by separation of iron ore (Brostow, Lobland,
Pal, & Singh, 2008), coal (Pal, Sen, Karmakar, Mal, & Singh, 2008),
dye contaminants from textile industry (Klimaviciute, Riauka, &
Zemaitaitis, 2007; Pal, Ghosh, Sen, Jha, & Singh, 2009), contaminat-
ing metals (Tseng, Wu, & Juang, 1999) or bentonites (Levy, Garti,
& Magdassi, 1995). Cationic cellulose (CelquatR), cationic guar,
and amino-modified starches are large-scale commercial products
playing an important role in papermaking processes, as retention
aids, strength agents, or antimicrobial agents for packaging.
or N-(glycidyl)-N,N,N-trimethylammonium chloride (GTAC)
(QUAB®151), and modified polysaccharides were cellulose (Abbott,
Bell, Handa, & Stoddart, 2006; Song, Sun, Zhang, Zhou, & Zhang,
2008; Yan, Tao, & Bangal, 2009), microcrystalline cellulose (Antal
& Micko, 1992), hemicellulose (Ren, Sun, Liu, Lin, & He, 2007),
starch (Haack, Heinze, Oelmeyer, & Kulicke, 2002; Kavaliauskaite,
Klimaviciute, & Zemaitaitis, 2008; Pal, Mal, & Singh, 2005; Wang
& Xie, 2010), corn cob meal (Simkovic, Mlynar, & Alfoldi, 1992),
glycogen (Pal, Mal, & Singh, 2006), arabinoxylan (Köhnke, Brelid, &
Westman, 2009), tamarind kernel polysaccharide (Pal et al., 2009),
guar gum (Huang, Yu, & Xiao, 2007), chitosan (Sajomsang, Gonil,
& Tantayanon, 2009; Sajomsang, Tantayanon, Tangpasuthadol, &
Daly, 2009), konjak glucomannan (Yu, Huang, Ying, & Xiao, 2007),
maltodextrin (Loiseau, Imbertie, Bories, Betbeder, & De Miguel,
2002), or dextran (Thomas, Rekha, & Sharma, 2010). The modifi-
cation can proceed in homogeneous (solutions) or heterogeneous
media (suspensions). These reactions require alkaline solutions
(NaOH) which accelerate the etherification reaction, but determine
also an intensification of reagent inactivation by hydrolysis when
the amount of NaOH exceeded 0.4 mol/anhydroglucose unit (AGU)
(Kavaliauskaite et al., 2008), and in some cases can damage the
polysaccharides’ supramolecular structure or degrade the polymer.
Amphiphilic cationic polysaccharides share properties and
applications with their hydrophilic analogues, but have addi-
tional properties due to their surface activity and ability to
self-organize in micelle like aggregates. There is only one com-
Synthesis of quaternary ammonium groups containing
polysaccharides have been performed by using commercially
∗
Corresponding author at: “Petru Poni” Institute of Macromolecular Chemistry,
Bioactive and Biocompatible Polymers, Aleea Grigore Ghica Voda 41 A, 700487 Iasi,
Romania. Tel.: +40 232 260333; fax: +40 232 211299.
E-mail address: nichifor@icmpp.ro (M. Nichifor).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.06.027

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M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
new cationic amphiphilic polysaccharides prepared by this proce-
dure are highlighted as well.
mercial product, Quatrisoft LM-200 (Polyquaternium-24), based
on hydroxyethyl cellulose with 5 mol% N-(2-hydroxypropyl)-N-
dodecyl-N,N-dimethylammonium chloride groups, largely used as
additive for personal care products (Zhang et al., 2008) or as thick-
ening agent (Zhang, 2001). Brode, Kreeger, and Salensky (1995)
and Brode, Kawakami, Doncel, and Kemnitzer (1998) reported
the synthesis of double cationic cellulose derivatives containing
both hydrophilic and amphiphilic pendant cationic groups. Other
amphiphilic cationic polysaccharides were obtained by the quater-
nization of tertiary amino groups containing polysaccharides with
alkyl halides (Souguir, Roudesli, About-Jaudet, Picton, & Le Cerf,
2010).
The present work describes the synthesis of polysaccharides
with quaternary ammonium pendant groups by one pot proce-
dure involving the reaction of a neutral polysaccharide (dextran,
pullulan, hydroxyethyl cellulose) with a mixture of epichlorohy-
drin (ECH) and a tertiary amine (R1R2R3N), where R1, R2, R3, are
identical or different, and at least one of the R substituents is an
alkyl chain with 2–16 carbons or a benzyl group. Heterocyclic ter-
tiary amines can be also used. The procedure can be applied to
various water soluble or water swellable polysaccharides in linear
or crosslinked forms. The reaction mechanism for dextran quater-
nization, cationic polymer chemical structure, and optimal reaction
conditions are presented. Several potential applications of some
2. Experimental
2.1. Materials
Dextran (Dex-40) with molar mass 40 000 was from S.C.
Sicomed S.A. (Bucharest, Romania). Pullulan (Pull-200) with a
molar mass of 200 000 was supplied by Hayashibara Lab, Okoyama,
Japan. All the other reagents were from Aldrich and were used
as received. The solvents were of analytical grade and were puri-
fied and/or dried by standard procedures. Crosslinked dextran gels
(CDex-w, as 0.2 mm spherical particles) were obtained as previ-
ously described (Nichifor, Cristea, Mocanu, & Carpov, 1998) and
are differentiated by their capacity for water retention (w), in gram
water/10 g gel. Thus, CDex-25 indicates a crosslinked dextran able
to retain 25 g water/10 g dry gel at equilibrium.
2.2. Synthesis of quaternization reagents
Synthesis of N-(3-chloro-2-hydroxypropyl)-N,N,N-trialkyl-
ammonium chloride (CHPR1R2R3A) was performed according
to the method described in Houben Weyl (1967), by reaction
Table 1
Tertiary amines and their derivatives used for polysaccharide quaternization.
R1R2R3N or R1R2R3R4N+Cl−
Code amine
DMEA
R1
R2
R3
R4
–
Method
iii
Maximum DS (a/AGU)a
CH3
CH3
C2H5
90 (10)
CHPDMEA
CH3
CH3
C2H5
i
50 (10)
GDMEA
TEA
CH3
CH3
C2H5
C2H5
ii
90 (10)
80 (10)
C2H5
C2H5
–
CHPTEA
GTEA
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
40 (10)
75 (10)
ii
DMBzA
CH3
CH3
iii
70 (9)
DMBuA
MDBuA
TBuA
DMOctA
MDOctA
DMDodA
DMCetA
CH3
CH3
C4H9
CH3
CH3
CH3
CH3
CH3
C4H9
C4H9
C4H9
C8H17
C8H17
C12H25
C16H33
–
–
–
–
–
–
–
iii
iii
iii
iii
iii
iii
iii
70 (9)
10 (8)
6 (8)
70 (10)
10 (10)
40 (4)
30 (6)
C4H9
C4H9
CH3
C8H17
CH3
CH3
GDMCetA
MeIm
CH3
CH3
C16H33
ii
5 (6)
–
–
iii
70 (9)
DABCO
iii
70 (8)
a
Maximum degree of substitution, in mol% (molar ratio of reagent mixture, a/AGU, where a refers to as amine reagents, necessary for obtaining this maximum DS). Reaction
conditions: 70 ◦C, 6 h, 1 g linear dextran/10 ml water.

M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
967
between R1R2R3N and ECH in water medium, with subsequent
addition of concentrated HCl till pH 3. The reagents were obtained
as 50% (w/w) water solutions. For analysis, the solid products were
separated by freeze drying.
(i) The reagent was CHPR1R2R3A (according to Table 1) (as
50 wt% water solution). Molar ratios AGU:reagent (active com-
pound):NaOH = 1:a:1.5a (here and in the following a refers to
as the amine reagents).
N-(3-chloro-2-hydroxypropyl)-N,N,N-triethylammonium chloride
(CHPTEA): Analysis, found (calculated), %: C 49.2 (47.2); H 9.71
(9.1); N 6.6 (6.1); total chlorine 24.0 (30.6); chloride ion 16.4 (15.3).
Active compound (calculated from covalent chlorine content, and
related to the solid product): 58%.
N-(3-chloro-2-hydroxypropyl)-N,N-dimethyl-N-ethylammonium
chloride (CHPDMEA): Found (calculated), %: C 43.7 (41.8); H 9.0
(8.4); N 7.6 (6.9); total chlorine 29.8 (34.8); chloride ion 19.3 (17.4).
Active compund: 60%.
(ii) The reagent was the glycidyl derivative of a tertiary
amine (GR1R2R3A). Molar ratios AGU:reagent (active com-
pound):NaOH = 1:a:0.5.
(iii) The reagent was an equimolar mixture of epichlorohydrin and
a tertiary amine (the molar ratio AGU:reagent mixture = 1:a).
When an excess of tertiary amine was used as a catalyst, the
molar ratios AGU:tertiary amine:ECH was 1:(a + 1):a.
Purification of quaternized products was performed as fol-
lows: crosslinked dextran gels were filtered and sequentially
rinsed on the filter with water, 0.1N NaOH, 0.1N HCl, water and
methanol. The final products were dried under vacuum. Aqueous
solutions of linear polysaccharides were precipitated in mixtures
methanol/acetone, then redissolved in deionized water and dia-
lyzed against 0.1N HCl using tubings with MW cut-off of 12–14 kDa
(Aldrich) then against water in an Amicon ultrafiltration cell
(Millipore) fitted with an Ultracel-PL-membrane with a nominal
molecular weight limit (NMWL) of 1 kDa, till the conductivity of the
dialyzate was close to that of deionized water. After concentration
in the same ultrafiltration cell, the solutions were freeze dried.
N-glycidyl-N,N,N-trialkylammonium chloride (GR1R2R3A)
were prepared from the corresponding tertiary amines and ECH
by the method of McClure and Williams (1969), using anhydrous
acetone as a solvent. The gel-like products were separated by
solvent evaporation, trituration with diethyl ether (for removal of
unreacted amine and ECH) and dried under vacuum.
N-glycidyl-N,N,N-triethylammonium chloride (GTEA): Yield,
related to tertiary amine: 40%. Analysis, found (calculated), %: C
54.8 (55.8); H 10.4 (10.3); N 6.9 (7.3); total chlorine 18.2 (18.3),
chloride ion 17.4 (18.3); epoxy group (EP) 0.34 (0.52). Active
compound (calculated from EP content): 65.8%.
N-glycidyl-N,N-dimethyl-N-ethylammonium chloride (GDMEA):
Yield: 45%. Analysis, found (calculated), %: C 49.8 (50.7); H 9.7 (9.6);
N 8.7 (8.8); total chlorine 20.0 (21.4), chloride ion 19.5 (21.4); epoxy
group (EP) 0.46 (0.6). Active compound: 75.5%.
N-glycidy-N,N-dimethyl-N-cetylammonium chloride (GDMCetA):
Yield: 10%. Analysis, found (calculated), %: C 70.1 (69.7); H 12.5
(12.2); N 3.7 (3.9); total chlorine 8.7 (9.8), chloride ion 8.1 (9.8);
epoxy group (EP) 0.17 (0.27). Active compound: 61.5%.
In the following, the reagent content in the quaternization mix-
tures was related to the active compounds. The reagents used in
the present paper are included in Table 1.
2.4. Characterization and analysis
2.4.1. Analysis of the reaction mixtures
The reaction mechanism was studied by analysis of two sets of
reaction mixtures, A and B, consisting of deionized water (30 ml),
DMBuA (5.62 g, 55.5 mmol) and ECH (5.13 g, 55.5 mmol), without
(A) or with dextran (3 g, 18.5 meq AGU) (B). At given time intervals
aliquots were withdrawn from both reaction mixtures, accurately
weighed (0.4–0.5 g) and analyzed for chloride ion (Cli) and epoxy
group content. The obtained values were related to the initial con-
tent of chlorine and epoxy groups corresponding to ECH amount in
the reaction mixture. Aliquots of about 10 ml were also taken from
the reaction mixture B, at similar time intervals, and precipitated
in methanol/acetone. Precipitated polymer samples (products Bt,
where t indicate the time interval at which the sample was with-
drawn) were purified once more by the same procedure, and after
drying under vacuum they were analyzed for the content in nitro-
gen, Cli, and total chlorine. After 24 h, the reaction mixture A was
freeze dried, giving product A, and the quaternized polymer from
reaction mixture B was purified by dialysis and recovered by freeze
drying (product B). Both products (A and B) were analyzed by FT-IR
and NMR.
2.3. Polysaccharide quaternization
Quaternization reactions were performed in water medium
(ratio polysaccharide/deionized water = 1 g/10 ml), under con-
stant stirring. Different quaternization reagents and catalysts,
according to procedures (i)–(iii) (Scheme 1) were applied. The
amounts of NaOH used in the procedures (i) (1.5 mol/AGU) and
(ii) (0.5 mol/AGU) correspond to optimal conditions previously
reported for polysaccharides quaternization by these procedures
(Kavaliauskaite et al., 2008; Wang and Xie, 2010).
2.4.2. Analysis methods
Elemental analysis (C, H, N) was carried out by means of a
CHNS 2400 II Perkin Elmer analyzer. The total chlorine content was
determined by Schoninger’s method followed by potentiometric
titration with AgNO3, using an automatic TitraLab Radiometer 840.
Chloride ions were quantified by potentiometric titration. Epoxy
group content was determined using the procedure described by
Jay (1964), by titration of exactly weighed aliquots from the reac-
tion mixtures A or B (about 0.5 g) with perchloric acid, in the
presence of tetrabutylammonium iodide, in acetic acid as solvent.
The method was validated by titration of known amounts of ECH in
the presence of water (0.1–0.5 ml) and dextran (0.2–0.3 g) amounts
corresponding to their concentrations in the reaction mixture’s
aliquots.
Degree of substitutions determined from epoxy group analysis
(DSEp) of the reaction mixtures A and B, and elemental analysis (N
or Cli) (DSEl) of the products Bt, were calculated with the following
Scheme 1. Procedures for the synthesis of polysaccharides with N-(2-
hydroxypropyl)-N,N,N-trialkylammonium chloride groups.

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M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
equations:
DSEp = a · {[Ep]A − [Ep]B}, mol/100 AGU
162 · El
100, mol/100 AGU
(1)
(2)
DSEl
=
100 · MEl − El · MS
where a is the molar ratio ECH/AGU, [Ep]A and [Ep]B are the residual
contents in epoxy groups, as % of the initial ECH content in the reac-
tion mixture of experiment A and B, respectively; El is the element
amount, in %, determined by elemental analysis (N) or potentio-
metric titration (Cli), MEl is elemental mass (14 for N, 35.5 for Cli),
and MS is the molecular weight of N-(2-hydroxypropyl)-N,N,N-
trialkylammonium chloride group. There was a good agreement
between DS determined from N and Cli content, therefore we used
in the following only DSCli. The DS is expressed as mol% in order
to avoid too many digits. For example, a DS value of 30 mol%
corresponds to 0.3 value used in the conventional notation of
polysaccharide substitution.
1H NMR and 13C NMR spectra were acquired on a Bruker Avance
DRX 400 spectrometer in D2O or DMSO-d6.
FT-IR spectra were recorded on a Bruker Vertex 70 spectrometer
using KBr pellets.
2.5. Antimicrobial activity assessments
In vitro antimicrobial activity of cationic dextran derivatives
obtained by procedure (iii) was assessed against three strains of
bacteria (Escherichia coli ATCC 22923, Staphylococcus aureus ATCC
25923, Pseudomonas aeruginosa ATCC 27835) and one fungal strain
(Candida albincas 90028). Before testing, pure cultures were real-
ized with all the strains in Mueller Hinton Agar. Sterile Petri dishes
(d = 10 cm) were prepared with a base layer of Mueller Hinton Agar
(Difco). Bacteria at density of 106–108 cfu were inoculated on solid
agar, and the Petri dishes were incubated at 37 ◦C for 24 h. The inoc-
ula were prepared by adjusting the turbidity of the suspension to
match the Mc Ferland standard 0.5 for bacteria and 2 for fungus.
The diluted microorganism cultures on Petri dishes were divided
in 4 zones and different amounts of polymer stock solution (con-
centration 200 ␮g/ml) were added to each zone, then incubated at
37 ◦C for 14 h. After this period, the results were visually assessed
by inhibition area.
Fig. 1. 1H NMR spectra (D2O) of Dex-40 modified with mixtures ECH/N-methyl imi-
dazol (a) and ECH/DABCO (b). Reaction conditions: 1 g polymer/10 ml water, molar
ratio reagents/AGU = 3/1, 70 ◦C, 6 h.
We reinvestigated the potential of the quaternization pathway
(iii) by using only one tertiary amine (the reagent) in the reac-
tion mixture, together with ECH, and tried to establish the possible
mechanism for polysaccharide substitution and to optimize reac-
tion conditions in order to obtain a DS as high as possible. The main
goal of the present study was to apply this procedure in the synthe-
sis of cationic amphiphilic polysaccharides with a well controlled
chemical structure. The polysaccharide of choice was dextran, and
we used either water soluble (linear) polymer for the study of the
reaction mechanism and confirmation of the chemical structure,
or crosslinked polymers for the comparative studies and reaction
parameters’ investigation.
3. Results and discussion
Scheme 1 presents the main procedures reported for the syn-
thesis of quaternary ammonium group containing polysaccharides.
Procedures (i) and (ii) commonly employ CHPTAC and GTAC
reagents, respectively, and the DSs depend on reaction conditions
and type of polysaccharide, ranging form 0.1 to 1.5 (10–150 mol%).
The use of a mixture tertiary amine/ECH (iii) for the surface
quaternization of cellulose was first reported by Gruber and Ott
(1996) who used two basic catalysts, 1-methyl-imidazol (MeIm)
and 1,4-diazabicyclo [2,2,2]octan (DABCO), and mentioned the lack
of reactivity of the mixture in the absence of these catalysts. The
DSs obtained by this procedure were very low (0.1–0.12 mmol/g, or
1.5–2 mol%).
When we first attempt to apply the procedure (iii) for polysac-
charide quaternization in homogeneous medium, we found that
the tertiary amines proposed as catalysts react themselves, at sig-
nificant levels, with polysaccharides, as 1H NMR spectra taken
for dextran modified with equimolar mixtures MeIm/ECH or
DABCO/ECH indicated the presence of specific signals for quater-
nized MeIm (Fig. 1a) or DABCO (Fig. 1b). Consequently, the use of
these “catalysts” together with another tertiary amine will result in
a cationic polysaccharide with a mixed and uncontrolled chemical
composition.
3.1. Mechanism of quaternization reaction by one pot procedure
In order to investigate the quaternization mechanism for pro-
cedure (iii), experiments A and B were performed as described in
Section 2. Experiment A was performed in the absence of dextran
and quantification of the chloride ions and epoxy groups at differ-
ent time intervals (Fig. 2a) indicated that, after 30 min at 40 ◦C, 80%
of the covalent chlorine (found in the initial amount of ECH) was
converted to chloride ions, but epoxy groups remain unchanged
(100% from initial), showing that the reaction between ECH and the
tertiary amine (NR3) leads mainly to the formation of the glycidyl
derivative 3 (Scheme 2a). At longer periods (>30 min), the chlo-
ride ion content increases, but with a lower rate, and reaches 94%
after 3 h. The occurrence of chloride ions in the reaction mixture
is mainly the result of reaction (a), and not to the ECH hydrolysis,
as an experiment performed under the same conditions but only

M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
969
Fig. 2. Time dependence of chloride ions and epoxy group contents in the reaction mixtures ECH/DMBuA/water (a) and ECH/DMBuA/Dex-40/water (c); and (b and d) degree
of substitution of Dex-40 with N-(2-hydroxypropyl)-N,N-dimethyl-N-butyl ammonium chloride groups as determined from epoxy group content in the reaction mixtures
or chloride ions in the separated and purified polymer samples (Bt). Content in covalently bound chlorine is also shown. Reaction conditions: 1 g polymer/10 ml water, molar
ratio reagents/AGU = 3/1, 40 ◦C (a and b) or 70 ◦C (c and d).
with ECH in the reaction medium did not reveal a significant con-
version of chlorine to chloride ions, which was less than 2% after
24 h at 40 ◦C. In the same time, epoxy content starts to decrease
slowly, perhaps by hydrolysis according to the reactions (b1) and
(b2) (Scheme 2), with formation of inactive glycidol derivatives.
The formation of glycidyl derivative 3 and its hydrolysis to gly-
cidol derivative 5 is supported by the 1H NMR spectrum of the
product (A), obtained by freeze drying of reaction mixture A, after
24 h at 40 ◦C. The spectrum (Fig. 3) indicates the presence of both
glycidyl and glycidol derivative specific peaks, and the content in
glycidyl derivative 3, calculated from the ratio of integrals for peaks
assigned to CH2 protons of the epoxy group (ı = 2.71 and 2.93 ppm)
and those for butyl chain protons CH3–CH2–CH2– (ı = 0.937, 1.306
and 1.68 ppm, respectively), was about 60%. This value fairly agrees
with the epoxy group content (67%) determined directly from the
reaction mixture A analysis, before freeze drying.
In the presence of dextran, the evolution of chloride ion con-
tent with time is very close to that found in the absence of dextran,
indicating that the reaction (a) proceeds with a similar rate with
formation of glycidyl derivative 3. However, epoxy group content
decreases faster in the presence of dextran, and we can assume that,
besides reaction (b), reactions (c) take place, with final formation of
polysaccharide derivative 6. If we assume that the reactions (b) pro-
ceed with the same rate irrespective of the dextran presence, the
difference between the epoxy group content in the presence and
the absence of dextran should represent the degree of substitution
in the product 6. Therefore, we compared DSEp calculated from the
difference in epoxy groups between experiments A and B (Eq. (1))
and DSCli determined from chloride analysis performed on prod-
ucts Bt (Eq. (2)). As Fig. 2b shows, the two DS values are very close,
with DSEp exceeding DSCli by about 1–2 mol%, and this difference
could arise from a lesser rate of 6 formation from 7. This hypothe-
sis was confirmed by the presence of covalent chlorine (1–2 mol%)
in the quaternized samples Bt separated from the reaction mixture
by precipitation. This chlorine disappeared at longer reaction times
(24 h), indicating either its hydrolysis or the reaction with tertiary
amine according to reaction (c2).
Similar experiments carried out at 70 ◦C (Fig. 2c and d) showed
an identical evolution of chloride ion content (data not shown)
(rapid formation of compound 3), and a faster rate of both quater-
nization reactions (c) and secondary reactions (b). A DS = 20 mol%
was obtained after 1.5 h at 70 ◦C and only after 8 h at 40 ◦C (Fig. 2b
and d). After 10 h in the absence of the polysaccharide, epoxy group
residual content was about 67% at 40 ◦C and 35% at 70 ◦C. It is worth
observing the lack of a difference between DSEp and DSCli (Fig. 2d)
meaning that the product 7 is not formed, or reaction (c2) goes
to completion, and this is supported by lower content in covalent
chlorine found in the polymer samples separated from the reac-
tion mixture when the reaction was performed at 70 ◦C (<0.5 mol%,
which falls into the experimental errors). The faster rate of sec-
ondary reactions (b) leads to a rapid leveling of the DS, which did
not overcome 25 mol% after 24 h, comparing with 36 mol% in case
of reaction performed at 40 ◦C.
3.2. Chemical structure of the quaternized products obtained by
one pot synthesis
A sample obtained by modification of dextran D-40 with a mix-
ture DMBuA/ECH, having a DS = 36 mol% (product B), was analyzed
by FT-IR, 1H NMR and 13C NMR. FT-IR spectrum showed, besides the

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M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
Scheme 2. Mechanism of polysaccharide quaternization in the presence of equimolar mixtures epichlorohydrin/tertiary amine.
bands characteristic for a polysaccharide, a clear band at 1486 cm−1
,
Kettenes-van den Bosch, van der Kerk-van Hoof, & Hennink, 1997)
and quaternary ammonium groups (Haack et al., 2002; Song et al.,
2008), a proof for chemical structure depicted in Fig. 4 and reaction
mechanism given in Scheme 2. The 13C NMR spectrum highlights
the presence of two new signals for saccharide ring carbons (not
found for unsubstituted dextran), at 96.3 ppm, assigned to the C1
adjacent to a substituted C2 (1ꢀS), and at 80 ppm for substituted
C2 (2S) (Heinze, Haack, & Rensing, 2004; van Dijk-Wolthuis et al.,
1997). No clear evidence for signals attributed to substituted C3
or C4 could be found, suggesting that, at the moderate DS of that
sample, the substitution takes place preferentially at C2–OH. For
much higher DS (about 90 mol%), the intensities of the signals cor-
responding to 1ꢀS and 2S (inset to Fig. 4b) are similar, indicating that
only about a half of ammonium groups are bound to C2, the other
being, most probably, located at C3 of dextran saccharide ring.
The ratio of the integrals of the signals assigned to CH3–(CH2)3–
(0.923 ppm) and anomeric glucopyranosidic protons (4.68 ppm)
(Fig. 4a) gives a DS of 38 mol%, close to the value determined from
elemental (N) and potentiometric (Cli) analysis (36 mol%).
which was attributed to the methyl groups bound to quaternary
nitrogen, and the band at 1420 cm−1 was referenced as the C–N
stretching vibration (Pal, Sen, et al., 2008; Song et al., 2008).
The NMR spectra of the product B clearly indicate the presence
of the signals characteristic to protons (Fig. 4b) and carbons (Fig. 4b)
of both saccharide rings (Bajgai et al., 2009; van Dijk-Wolthuis,
3.3. Comparison of quaternization procedures
It is well established that the modification of polysaccharides
with CHPTAC in the presence of NaOH (procedure i) takes place
actually via GTAC, which is formed in situ from CHPTAC and NaOH
(Heinze et al., 2004; Kavaliauskaite et al., 2008). We have already
shown that the glycidyl derivatives are the reagents in the proce-
dure (iii) too. The comparative efficiency of the three procedures
Fig. 3. 1H NMR spectrum (DMSO-d6) of reaction mixture ECH/DMBuA/water freeze
dried after 24 h at 40 ◦C (product A).

M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
971
Fig. 4. NMR spectra (DMSO-d6) of Dex-40 with N-(2-hydroxypropyl)-N,N-dimethyl-N-butyl ammonium chloride groups. DS = 36 mol%. Inset to (b) DS = 90 mol%.
is illustrated in Fig. 5, using as starting reagents triethylamine
(TEA)/ECH mixture and clorohydroxypropyl or glycidyl derivatives
of TEA. In all cases, the DS increases with increase in the ratio a/AGU,
but the efficiency decreases in the order: (ii) > (iii) > (i). The higher
efficiency of GTAC versus CHPTAC was previously shown for the
starch quaternization (Bendoraitiene, Kavaliauskaite, Klimaviciute,
& Zemaitaitis, 2006; Haack et al., 2002), and was explained by the
competition between the main quaternization reaction and the
CHPTAC enhanced inactivation due to the higher content in NaOH
required for the procedure (i). The better performances of the pro-
cedure (iii) in comparison with (i) can be a supplementary proof of
glycidyl derivative formation in situ, in the absence of NaOH.
The quaternization efficiency proved to be better for (ii) pro-
cedure only for tertiary amines with short substituents (mainly
CH3). When amines with longer alkyl substituents were used (for
instance TEA of DMCetA), the better results obtained by procedure
(iii) are obvious (Fig. 5b). The explanation could be either the contri-
bution of reaction (c2) (Scheme 2) to the final substitution efficiency
or a better reagent compatibility/solubilization of the step-wise
formed glycidyl derivatives of more hydrophobic tertiary amines.
3.4. Factors influencing the efficiency of quaternization reaction
Several factors are responsible for quaternization efficiency by
procedure (iii): temperature, catalyst, solvent, hydrophobicity or
bulkyness of the amine substituents, and polysaccharide support
characteristics.
The influence of the temperature on the reactions (c) efficiency
was presented above. Higher reaction temperatures decrease the
reaction time but diminish the final DS, therefore the proper choice
of experimental temperature will depend on the desired cationic
polymer characteristics and reagent properties (tertiary amine
boiling point, polysaccharide thermal resistance).
In the absence of any catalysts and at equimolar ratio tertiary
amine/ECH, satisfactory DS were obtained by procedure iii, compa-
rable with those of the other procedures. The presence of NaOH in

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M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
(c) (Scheme 2) completion. Actually, experiments performed with
mixtures of MDBuA or MDOctA and ECH in water solution, in the
absence of polysaccharide, have shown that, after 6 h at 70 ◦C, large
amounts of unreacted amines (1/4 of MDBuA and 1/2 of MDOctA
used in experiments) were separated from the reaction mixtures.
The maximum DS, i.e. the highest possible DS for each tertiary
amine used as reagent, is indicated in Table 1 (last column). The
highest DSs given for more hydrophobic amine reagents are leveled
by the requirement of water solubility (or swellability) preserva-
tion for quaternized polysaccharides.
The reactivity of cyclic amines MeIm and DABCO was similar to
acyclic amines with shorter alkyl substituents (DMBuA). It is worth
mentioning that the quaternized polysaccharide obtained in the
presence of DABCO as reagent (DS = 40 mol%) was water soluble,
showing that only one tertiary nitrogen is involved in the prepa-
ration of glycidyl derivative 3 (Scheme 2) what was supported by
1H NMR spectrum (Fig. 1b) where the protons of both CH2 groups
bound to tertiary nitrogen (ı = 3.25 ppm) and quaternary nitrogen
(ı = 3.4–3.6 ppm) are present.
Fig. 6b illustrates the influence of the polysaccharide support
properties on DS. The DS of a linear dextran was higher than that of
crosslinked dextran, and a higher crosslinking degree (or a lower
water retention capacity) led to a lower DS. This effect is obviously
the result of a reduced diffusion of reagents inside the polysaccha-
ride gels, which decreases with increasing crosslinking degree. As
for the polysaccharide type, the quaternization efficacy was higher
for pullulan than dextran (both as linear polymers), what is clearly
due to the higher reactivity of the primary OH groups present in
the pullulan structure. Molecular mass of the linear polysaccharide
had no significant influence on the DS (data not shown).
3.5. Potential applications of amphiphilic quaternary ammonium
group containing polysaccharides
Using procedure (iii), we have prepared
a large variety
of cationic hydrophilic or amphiphilic polysaccharides, with a
well controlled and reproducible chemical structure, and their
amphiphilicity could be easily varied by changing the length
of a single substituent at nitrogen (R3, according to Table 1).
Therefore, we synthesized cationic amphiphilic dextran derivatives
(crosslinked or as water soluble products) with different R3 sub-
stituents at nitrogen (R3 = C2, C4, C8, C12, C16) and studied their
properties and potential for different applications. The chemical
composition of the dextran derivatives discussed in the follow-
ing is indicated by the tertiary amine used for their preparation
(according to Table 1).
Fig. 5. Comparison of the efficacy of quaternization procedures, for the quaterniza-
tion of crosslinked dextran (CDex-25) as a function of (a) molar ratio reagents/AGU
and catalyst; (b) the length of amine substituents (molar ratio reagents/AGU = 3/1).
Reaction conditions: 70 ◦C, 6 h, 1 g polymer/10 ml water. (iii) NaOH (or TEA) indi-
cates that the reaction was performed with 1 mol catalyst/AGU, where the catalyst
was NaOH (or TEA). DS was calculated with Eq. (2).
Cationic amphiphilic derivatives of crosslinked dextran were
tested in vitro for the sorption of sodium salts of bile acids (Nichifor,
Zhu, Baille, Cristea, & Carpov, 2001; Nichifor, Zhu, Cristea, & Carpov,
2001), in the attempt to find the best chemical structure for their
use as bile acid sequestrants, a well known class of anticholes-
terolemic drugs. The interaction of the steroid nucleus with one
amphiphilic gel (DMDodA) was stronger in comparison with the
gels having aromatic rings either as a part of polymer backbone
(Cholestyramine®, a classic anticholesteremic drug) or as a sub-
stituent of the functional group (DMBzA). No improvement was
noticed in the bile acid affinity for gels obtained from cyclic amines
(MeIm or DABCO). The better performances of the amphiphilic
cationic dextran gel (DMDodA) by comparison with Cholestyra-
mine were latter confirmed by in vivo experiments performed on
normolipemic rats, indicating such gels as promising hipolipemic
drugs, with better therapeutic activity and better tolerance than
Cholestyramine (Trinca, Nichifor, Volf, Ivas, & Stanciu, 2007).
The ability of water soluble cationic amphiphilic dextrans to
self-aggregate in water solutions with formation of intra- or
inter-molecular hydrophobic microdomains was demonstrated
the reaction mixture (1 mol/AGU) did reduce the reaction efficiency
(Fig. 5a, curve 2). However, an excess of the tertiary amine in the
reaction mixture (molar ratio amine/ECH = (a + 1)/a), significantly
improves the reaction efficiency (Fig. 5a, curve 6). A similar behav-
ior was noticed for the procedure (ii) when instead NaOH, TEA was
used (curve 5 in Fig. 4). This suggests that the tertiary amine play
the role of the catalyst in the reaction (c1).
The best solvent for the polysaccharide quaternization by pro-
cedure (iii) was water. Very low DSs were obtained when other
solvents (DMSO, N-methylformamide, DMF) or their mixtures with
water were used. The main reason for this solvent specificity might
be the low rate of the reaction (a) in these solvents, as compared
with water.
The length of the alkyl substituents at the nitrogen of tertiary
amine used as a reagent also influences DS, as Fig. 6a shows. The
reaction efficiency decreases with increasing R3 length when ter-
tiary amines DMR3A were used as reagents. Introduction of two
or three longer alkyl substituents determines a further decrease
in DS, clearly due to the steric hindrance in the reactions (a) and

M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
973
Fig. 6. Quaternization efficacy performed with procedure (iii) as a function of the tertiary amine substituent length (for polymer = CDex-25) (a); and polysaccharide support
(for DMDodA) (b). Reaction conditions 1 g polymer/10 ml water, 70 ◦C, 6 h. DS was calculated with Eq. (2).
Table 2
by an extensive fluorescence study (Nichifor, Lopes, Bastos, &
Antibacterial and antifungal properties of quaternary ammonium group containing
dextran.
Lopes, 2004), and by isothermal titration calorimetry (Bai, Nichifor,
Lopes, & Bastos, 2005b). These amphiphilic polymers interact
with surfactant of opposite charge (Bai, Santos, Nichifor, Lopes,
& Bastos, 2004; Bai, Nichifor, Lopes, & Bastos, 2005a; Nichifor,
Polymera
Microorganismb
E. coli
S. aureus
P. aeuruginosa
C. albicans
Lopes, Bastos,
& Lopes, 2008) or of the same charge (Bai,
DMEA 30
DMEA 70
−
−
−
−
+
n.d.
n.d.
n.d.
n.d.
+
+
++
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
+
++
++
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
−
Catita, Nichifor, & Bastos, 2007) with formation of mixed aggre-
gates polymer-surfactant. Both polymer self-aggregates and mixed
polymer-surfactant aggregates are able to solubilize hydrophobic
compounds and, therefore, can be applied as hydrophobic drug
delivery systems for oral or systemic administration.
Water soluble cationic dextran derivatives, both hydrophilic
and amphiphilic, proved themselves as good flocculants for clay
suspensions (Ghimici, Morariu, & Nichifor, 2009). The optimum
flocculation dose and the width of the flocculation window were
strongly dependent on DS and length of substituent R3.
DMBuA 30
DMBuA 70
DMBzA 30
DMOctA 30
DMOctA 70
DMDodA 30
DMCetA 30
+
−
++
−
−
−
n.d.
n.d.
a
Polymers were identified after the tertiary amine used in the synthesis (accord-
ing to Table 1) and the DS, as mol quaternary ammonium group/100 AGU.
b
‘−’ no inhibition; ‘+’ inhibition; ‘++’ strong inhibition; ‘n. d.’ not determined.
Another potential application of cationic amphiphilic polysac-
charides is as antibacterial agents. Quaternary ammonium
compounds are well known antibacterial agents used for disinfec-
tion, and antibacterial properties of several quaternary ammonium
group containing polysaccharides have been reported (Sajomsang,
Gonil, et al., 2009; Sajomsang, Tantayanon, et al., 2009; Yu et al.,
2007). A first trial performed on E. coli showed that only polymers
DMBzA and DMOctA were active, meaning that only polymers car-
rying groups with a medium length of substituent R3 (benzyl and
octyl) are able to interact with the bacteria cell membrane. All
other polymers having short (ethyl, butyl) and long (dodecyl, cetyl)
alkyl substituents at quaternary ammonium groups did not display
any antibacterial activity (Table 2). The DMBzA and DMOctA poly-
mers were further tested for their activity against other bacteria
(P. aeruginosa and S. aureus) and a fungus (C. albicans). No activ-
ity against fungus was found, but the tested polymers were active
against bacteria strains studied, and inhibition activity increased in
the order: DMBzA 30 < DMOctA 30 < DMOctA 70 (Fig. 7), suggest-
Fig. 7. Images of the E. coli plates incubated with cationic dextran derivatives (polymer names as in Table 2). The amount of polymer stock solution was 10 ␮l (zone 1); 20 ␮l
(zone 2); ␮l (zone 3); 0 (zone 4).

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M. Nichifor et al. / Carbohydrate Polymers 82 (2010) 965–975
ing a better performance of polymers carrying an octyl substituent
and an increase in activity with increasing DS. A study focused on
elucidation of the reason for different activity decided by only one
substituent at quaternary ammonium groups and also on quan-
tification of this activity (minimum inhibitory concentration-MIC
determination) is in progress.
Ghimici, L., Morariu, S., & Nichifor, M. (2009). Separation of clay suspen-
sion by ionic dextran derivatives. Separation and Purification Techniques, 68,
165–171.
Gruber, E., & Ott, T. (1996). Eine neue Methode zur Herstellung von kationischen
Derivaten von Polysacchariden. Das Papier, 4, 157–162.
Haack, V., Heinze, T., Oelmeyer, G., & Kulicke, W. M. (2002). Starch derivatives
of high degree of functionalization, 8. Synthesis and flocculation behavior of
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495–502.
Heinze, T., Haack, V., & Rensing, S. (2004). Starch derivatives of high degree of func-
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4. Conclusion
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A new versatile procedure for synthesis of quaternary ammo-
nium group containing polysaccharides was described. Instead of
preformed quaternary ammonium group containing reagents, the
procedure uses a mixture of a tertiary amine and epichlohydrin
which give rise, in situ, to the quaternary ammonium reagents.
This procedure avoids the difficult and expensive synthesis of qua-
ternary ammonium reagents and allows the obtaining of a large
number of polysaccharide derivatives with different properties and
various applications, simply by changing the tertiary amine used in
reaction. The method can be applied to the quaternization of any
neutral polysaccharide, in linear or crosslinked form, and lead to
the formation of well controlled chemical structures, with pendant
quaternary ammonium groups statistically distributed along the
polysaccharide chain.
The procedure allowed the preparation of polysaccharides with
different hydrophilic–lipophilic balance by simply changing the
length of one substituent at the tertiary amine used as a reagent.
These new cationic polysaccharides have self-assembling proper-
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