Impact of chain length on antibacterial activity and hemocompatibility of quaternary N-alkyl and N, N-dialkyl chitosan derivatives

Article abstract of DOI:10.1021/acs.biomac.5b00163

A highly efficient method for chemical modification of chitosan biopolymers by reductive amination to yield N,N-dialkyl chitosan derivatives was developed. The use of 3,6-O-di-tert-butyldimethylsilylchitosan as a precursor enabled the first 100% disubstitution of the amino groups with long alkyl chains. The corresponding mono N-alkyl derivatives were also synthesized, and all the alkyl compounds were then quaternized using an optimized procedure. These well-defined derivatives were studied for antibacterial activity against Gram positive S. aureus, E. faecalis, and Gram negative E. coli, P. aeruginosa, which could be correlated to the length of the alkyl chain, but the order was dependent on the bacterial strain. Toxicity against human red blood cells and human epithelial Caco-2 cells was found to be proportional to the length of the alkyl chain. The most active chitosan derivatives were found to be more selective for killing bacteria than the quaternary ammonium disinfectants cetylpyridinium chloride and benzalkonium chloride, as well as the antimicrobial peptides melittin and LL-37.

Full text of DOI:10.1021/acs.biomac.5b00163

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Article
The Impact of Chain Length on Antibacterial Activity and Hemocompatibility
of Quaternary N-Alkyl and N,N-Dialkyl Chitosan Derivatives
PRIYANKA SAHARIAH, Berglind Eva Benediktsdóttir, Martha A. Hjálmarsdóttir, Olafur Eysteinn
Sigurjonsson, Kasper K. Sørensen, Mikkel Boas Thygesen, Knud Jørgen Jensen, and Már Másson
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00163 • Publication Date (Web): 01 Apr 2015
Downloaded from http://pubs.acs.org on April 6, 2015
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The Impact of Chain Length on Antibacterial
Activity and Hemocompatibility of Quaternary Nꢀ
Alkyl and N,NꢀDialkyl Chitosan Derivatives
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Priyanka Sahariaha, Berglind E. Benediktssdóttira, Martha Á. Hjálmarsdóttirb, Olafur E.
Sigurjonssonc,d, Kasper K. Sørensene, Mikkel B. Thygesene, Knud J. Jensene, Már Mássona,*s
aFaculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland,
Hofsvallagata 53, ISꢀ107 Reykjavik, Iceland
bDepartment of Biomedical Science, Faculty of Medicine, University of Iceland, Stapi,
Hringbraut 31, 101 Reykjavik, Iceland
cThe REModeL Lab, The Blood Bank, Landspitali University Hospital, Snorrabraut 60, 105
Reykjavik, Iceland
dInstitute of Biomedical and Neural Engineering, Reykjavik University, Menntavegur 1, 101,
Reykjavik, Iceland
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eDepartment of Chemistry, Faculty of Science, Centre for Carbohydrate Recognition and
Signalling, University of Copenhagen, Thorvaldsensvej 40, DKꢀ1871 Fredriksberg C,
Copenhagen, Denmark
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Keywords: 3,6,ꢀOꢀdiTBDMS chitosan; Nꢀalkyl chitosan; Quaternary chitosan; Antibacterial
activity; Hemolytic activity; Structureꢀactivity relationship
Abstract
A highly efficient method for chemical modification of chitosan biopolymers by reductive
amination to yield N,Nꢀdialkyl chitosan derivatives was developed. The use of 3,6ꢀOꢀdiꢀtertꢀ
butyldimethylsilylchitosan as a precursor enabled the first 100% disubstitution of the amino
groups with long alkyl chains. The corresponding mono Nꢀalkyl derivatives were also
synthesized, and all the alkyl compounds were then quaternized using an optimized procedure.
These wellꢀdefined derivatives were studied for antibacterial activity against Grampositive
S.aureus, E.faecalis and Gramnegative E.coli, P.aeruginosa which could be correlated to the
length of the alkyl chain, but the order was dependent on the bacterial strain. Toxicity against
human red blood cells and human epithelial Cacoꢀ2 cells was found to be proportional to the
length of the alkyl chain. The most active chitosan derivatives were found to be more selective
for killing bacteria than the quaternary ammonium disinfectants cetylpyridiniumchloride and
benzalkoniumchloride as well as the antimicrobial peptides melittin and LLꢀ37.
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1. INTRODUCTION
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Chitin represent a class of linear polysaccharides comprised of NꢀacetylꢀDꢀglucosamine units
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linked together with βꢀ(1–4) glycosidic bonds. Chitin is the second most abundant biopolymer in
nature.1,2 It can be isolated from the exoskeleton of crustaceans such as shrimps and crabs, as
well as from the cell walls of fungi.3 Chemical deacetylation of chitin can be applied to produce
the highly deacetylated form, which is named chitosan. Chitosan contains three distinct
functional groups, that is, one primary amino group and two hydroxyl groups (primary and
secondary), all of which can be synthetically modified to obtain derivatives with improved
biological properties. The quaternary derivatives of Nꢀalkyl chitosan have been shown to be
effective as antimicrobial agents,4ꢀ6 apart from finding application in the field of tissue
engineering,7 DNA delivery,8 drug delivery,9 and membrane coating.10
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The influence of the cationic charge and the chemical properties of the hydrophobic groups on
the antimicrobial activity of chitosan derivatives has been the subject of several studies.5,6
Rúnarsson et al. found11 that derivatives of chitosan containing quaternary ammonium moieties
like Nꢀalkyl or pyridinium groups had enhanced antibacterial activity compared to native
chitosan. Surfaceꢀquaternized derivatives of chitosan carrying high positive charge density and
containing hydrophobic Nꢀsubstituents like benzyl moieties were shown to inhibit the growth of
Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) more strongly than derivatives
containing Nꢀpropyl groups.5 On the other hand, Vallapa et al. observed a decreasing inhibitory
effect on S.aureus and E.coli as the hydrophobicity increased from ethyl to butyl and then to
benzyl.6 Although the maximum efficacy was observed with highly hydrophobic moieties and
long alkyl chain lengths in some studies,4 others found optimum activity with moderate chain
length.12 This discrepancy in the results may be attributed to the significant variability in the
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reported degree of substitution (DS) in the products synthesized for these studies. Selective
synthesis that allows good control of the DS is, therefore, required to gain a detailed
understanding of the structure–activity relationships of these materials.
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Reductive alkylation has been used for the introduction of alkyl groups to the amino group of
chitosan.13 This reaction is often preferred to substitution reactions with alkyl halides, since
reductive alkylation is Nꢀselective and results in monoꢀ or dialkyl derivatives, avoiding
quaternization and, hence, it gives some control over the synthesis. However, such synthetic
modification on native chitosan can be difficult owing to the insolubility of the biopolymer in
aqueous medium or organic solvents. The synthesis of Nꢀalkyl chitosan derivatives which has
usually been carried out in acidic14 or heterogeneous medium (1:1 water:ethanol)15 have not led
to 100% dialkylation through reductive amination with groups other than methyl.14 This has
proven to be particularly difficult with the longer alkyl chains. Even the use of excess reagents
(11–15ꢀfold excess) under aqueous conditions has only led to partial dialkylation with butyl
chains (DS = 0.5) and monoalkylation with octyl (DS = 0.9) and dodecyl chains (DS = 0.9).16
Other studies have reported even lower DS.10,17ꢀ19 The possible reason for this is that Nꢀ
alkylation tends to reduce the solubility of the polymer in aqueous medium, which may prevent
100% dialkylation with more hydrophobic moieties. Quaternization of such derivatives,
therefore, leads to a heteropolymer with partial quaternization of the Nꢀalkyl and N,Nꢀdialkyl
moieties. As the DS tends to vary with chain length, a comparison of the previously reported
structure–activity relationships should be interpreted with caution.
In the current study, reductive alkylation was carried out in organic solvent with the aid of
TBDMSꢀprotected chitosan (TBDMS = tertꢀbutyldimethylsilyl). TBDMS protection of the
hydroxyl groups gives rise to a precursor that has high solubility in a wide range of organic
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solvents. This, in turn, gives good control on the synthesis and aids in regioselective Nꢀ
modification.20,21 Recently, TBDMS protection has been utilized in reductive amination to yield
monoꢀNꢀalkylated chitosan derivatives with different chain lengths. Quaternization of these
alkylated products also resulted in highly substituted products.22 In general, it has been
concluded that hydrophobicity tends to increase antibacterial activity; however, a detailed study
of wellꢀcharacterized quaternary chitosan derivatives, showing the degree of hydrophobicity
required for optimum activity against various bacterial species, has not previously been reported.
Herein, we report the synthesis of seven different Nꢀalkyl quaternary chitosan derivatives,
differing in hydrophobicity (i.e. Nꢀalkyl substitution) and having a uniform degree of
quaternization. These compounds have been assessed against a panel of clinically relevant
bacterial strains under similar experimental conditions, so as to give a better understanding of the
structure–activity relationships of antimicrobial chitosan derivatives. Selectivity was assessed by
comparing the antimicrobial and hemolytic activity. Preliminary toxicity of the derivatives was
determined against colorectal adenocarcinomaꢀderived cell line, Cacoꢀ2. Furthermore, the
chitosan derivatives were compared to two quaternary ammonium disinfectants and two
antimicrobial peptides in order to gain a better overview of the antimicrobial action and their
suitability for clinical applications.
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2. EXPERIMENTAL
2.1. Materials. Chitosan polymer (S030626ꢀ2) was obtained from Genis ehf (Reykjavik,
Iceland). Its degree of deacetylation (DA) was found to be 94%, as obtained from the integrals of
1H NMR spectrum.23 The average molecular weight (Mw) of chitosan starting material was
calculated to be 294 kDa using size exclusion chromatography. All chemicals were purchased
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from Sigma–Aldrich and used without any further purification. Fmoc amino acids were
purchased from Iris Biotech GmbH. Dialysis membranes (RC, Spectra/Por, Mw cutoff 3500 Da)
and FloatꢀAꢀLyzers (Spectra/Por, Mw cutoff 3.5–5 kDa, 5 mL sample volume) were purchased
from Spectrum® Laboratories Inc. (Rancho Dominguez, USA). Staphylococcus aureus (ATCC
29213), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and
Pseudomonas aeruginosa (ATCC 27853) were obtained from the American Type Culture
Collection. Mueller–Hinton Broth and blood agar [heart infusion agar with 5% (v/v) defibrinated
horse blood] were purchased from Oxoid (Hampshire, UK).
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2.2. Characterization. Detailed characterization of the compounds was performed using H
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and COSY NMR and IR spectroscopy. H and COSY NMR spectra were recorded using a
Bruker Avance 400 instrument operating at 400.13 MHz at 300 K. NMR samples were prepared
in CDCl3, DMSOꢀd6, and D2O in concentrations of 10 to 15 mg/mL. The Nꢀacetyl peak (2.08
ppm)24 was used as the internal reference in all spectra. FTꢀIR measurements were performed
with an AVATAR 370 FTꢀIR instrument (Thermo Nicolet Corporation, Madison, USA) with 32
scans and a resolution of 4 cm−1 and a Specac compressor (Specac Inc., Smyrna, USA).
Equivalent quantities of the reagents were calculated on the basis of one glucosamine unit. The
DS for the Nꢀalkyl chitosan derivatives was evaluated on the basis of the integral values in the 1H
NMR spectra. The following equations were used to calculate the DS for the chitosan
derivatives:
∫ ꢁꢂꢃ
ꢆ
1) Degree of acetylation, DA = ꢀ∫ ꢁꢄꢅꢁꢆ ˣ ꢈ x 100
(1)
(2)
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ꢒ
3) DS for N,NꢀdialkylꢀNꢀmethyl, B = ꢀ∫ ꢓꢅꢉꢁ
∫ ꢁꢒ,ꢁꢒꢊ´ ˣ ꢈ x 100
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Where H1, H2 and H6 are the protons at position 1, 2 and 6 respectively in the glucosamine unit;
HAc are the protons in the Nꢀacetyl group; CH3 are the terminal protons in the alkyl chain; N–
CH3 are the protons in the methyl group; H1 and H1' in equation (3) represents proton at position
1 for the N,NꢀdialkylꢀNꢀmethyl and the N,Nꢀdialkyl units respectively.
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2.3. Synthesis. 2.3.1. 3,6ꢀOꢀdiTBDMS chitosan (1). 3,6ꢀOꢀdiTBDMS chitosan 1 was
synthesized via the chitosan mesylate salt, according to a previously published procedure.21
2.3.2. Synthesis of N,N,Nꢀtrimethyl chitosan (3). N,N,Nꢀtrimethyl chitosan was synthesized
based on a previously published procedure.22 Briefly, Cs2CO3 (4.63 g, 14.2 mmol) was added to
a solution of 1 (1.42 g, 3.6 mmol) in dry Nꢀmethylꢀ2ꢀpyrrolidone (NMP; 20 mL). After stirring
for 1 h, CH3I (1.11 mL, 17.8 mmol) was added dropwise under cooling and the reaction was
heated in a closed reaction vial at 50 oC for 48 h. The reaction mixture was purified by dialysing
against deionized water for 2 days. The solution was then freezeꢀdried to afford N,N,Nꢀtrimethylꢀ
3,6ꢀOꢀdiTBDMS chitosan iodide 2 (scheme 1) as a dark red product. Yield: 93%. Compound 2
(1.85 g, 3.30 mmol) was deprotected by treatment with 1 M tetra butyl ammonium fluoride
(TBAF) solution in NMP (10 mL) at 50 °C for 48 h. The resulting solution was dialyzed for 2
days against deionized water, then ionꢀexchanged with 10% NaCl (aqueous; w/v) overnight, and
this was then followed by dialysis against deionized water for another 2 days. The resulting
compound was then freezeꢀdried, resulting in light brown and “fluffy” N,N,Nꢀtrimethyl chitosan
chloride 3. Yield: 74%. 1H NMR (400 MHz, D2O): δ 2.08 (NCOCH3), 3.35 [N(CH3)3], 3.75 (Hꢀ
2), 3.90 (Hꢀ6), 3.99 (Hꢀ5), 4.36 (Hꢀ4), 4.47 (Hꢀ3), 5.49 (Hꢀ1) ppm. FTꢀIR (KBr): ν 3386 (O–H),
2930 (s, C–H), 1639 (C=O amide I), 1482 (s, C–H), 1052 (br s, C–O) cmꢀ1.
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2.3.3. Synthesis of NꢀalkylꢀN,Nꢀdimethyl chitosan derivatives (5a–c): General procedure.
The NꢀalkylꢀN,Nꢀdimethyl chitosan derivatives were synthesized according to a previously
developed procedure.22 Briefly, 3,6ꢀOꢀdiTBDMS chitosan 1 (1 g, 2.5 mmol) was dissolved in
CH2Cl2 (10 mL) and then stirred with aldehyde (12.7 mmol), triethylamine (0.38 mL, 2.75
mmol), and molecular sieves at 45 °C for 96 h. The reaction mixture was evaporated to dryness
and the resulting solid was washed with acetonitrile (15 mL), filtered, and airꢀdried. This
material was then reꢀdissolved in CH2Cl2 (10 mL) and stirred at room temperature with acetic
acid (0.57 mL, 10 mmol) and Na(OAc)3BH (2.1 g, 10 mmol) for 24 h. After completion, the
material was precipitated with acetonitrile (20 mL), filtered, and finally washed with water (10
mL), acetonitrile (10 mL) before being dried to give a yellow solid. The Nꢀalkylated material
was then quaternized by dissolving the material in CH2Cl2 (5 mL) in a sealed tube and adding
LiCO3 (0.73 g, 10 mmol) and dimethylsulfate (Me2SO4, 1.89 mL, 20 mmol), and the mixture was
heated at 50 °C for 48 h. After reaction completion, the mixture was dialyzed against water for 2
days and then freezeꢀdried for 2 days. The NꢀalkylꢀN,NꢀdimethylꢀdiTBDMS chitosan derivatives
4a–c were then deprotected using 1M TBAF/NMP solution (10 mL) at 50 °C for 48 h. The
resulting solutions were then dialyzed against deionized water for 2 days, ionꢀexchanged by
stirring in 10% NaCl (w/v) overnight, followed by dialysis against deionized water for 2 days.
The resulting compounds were then freezeꢀdried for 2 days, giving the NꢀalkylꢀN,Nꢀdimethyl
chitosan derivatives 5a–c (scheme 1) as light yellow solids. NꢀethylꢀN,Nꢀdimethyl chitosan
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(5a): H NMR (400 MHz, D2O): δ 1.44 (Hꢀ8), 2.08 (NCOCH3), 3.25 [N(CH3)2], 3.33 (N–CH3),
3.46–3.79 (Hꢀ2, Hꢀ7), 3.89–3.96 (Hꢀ5, Hꢀ6), 4.36 (Hꢀ4), 4.50 (Hꢀ3), 5.52 (Hꢀ1) ppm. FTꢀIR
(KBr): ν 3385 (O–H), 2956–2858 (s, C–H), 1637 (C=O amide I), 1479 (s, C–H), 1377 (C–H),
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1052 (br s, C–O) cmꢀ1. NꢀbutylꢀN,Nꢀdimethyl chitosan (5b): 1H NMR (400 MHz, D2O): δ 0.91
(Hꢀ10), 1.36 (Hꢀ9), 1.86 (Hꢀ10), 2.08 (NCOCH3), 3.27 [N(CH3)2], 3.34 (N–CH3), 3.47–3.9 (Hꢀ2,
Hꢀ7), 3.87–4.50 (Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6), 5.30–5.54 (Hꢀ1) ppm. FTꢀIR (KBr): ν 3423 (O–H), 2956–
2859 (s, C–H), 1630 (C=O amide I), 1466 (s, C–H), 1376 (C–H), 1056 (br s, C–O) cmꢀ1. Nꢀ
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hexylꢀN,Nꢀdimethyl chitosan (5c): H NMR (400 MHz, D2O): δ 0.95 (Hꢀ12), 1.40 (Hꢀ9, Hꢀ10,
Hꢀ11), 1.87 (Hꢀ8), 2.08 (NCOCH3), 3.27 [N(CH3)2], 3.5–3.78 (Hꢀ2, Hꢀ7), 4.37–4.49 (Hꢀ3, Hꢀ4,
Hꢀ5, Hꢀ6), 5.25–5.54 (Hꢀ1) ppm. FTꢀIR (KBr): ν 3419 (O–H), 2958–2873 (s, C–H), 1634 (C=O
amide I), 1464 (s, C–H), 1375 (C–H), 1055 (br s, C–O) cmꢀ1.
2.3.4. Synthesis of N,Nꢀdialkylꢀ3,6ꢀOꢀdiTBDMS chitosan derivatives (6a–d): General
procedure. To a solution of compound 1 (1g, 2.5 mmol) in CH2Cl2 (100 mL), aldehyde (5
equiv), acetic acid (0.5 mL), and molecular sieves were added, and the resulting mixture was
stirred for 24 h. Sodium borohydride (5 equiv) was then added slowly whilst cooling, and the
reaction mixture was left for another 24 h. The addition of aldehyde (5 equiv) and sodium
borohydride (5 equiv) was repeated once more. After an additional 24 h of stirring, the reaction
mixture was filtered, concentrated, and precipitated with acetone. The precipitated material was
then washed thoroughly with water (100 mL) and acetone (100 mL), and then dried to give N,Nꢀ
dialkylꢀ3,6ꢀOꢀdiTBDMS chitosan. N,Nꢀdimethylꢀ 3,6ꢀOꢀdiTBDMS chitosan (6a): Yield: 91%.
1H NMR (400 MHz, CDCl3): δ 0.05 [SiC(CH3)3], 0.87 [Si(CH3)2], 3.3 [N(CH3)2], 3.6–4.0 (Hꢀ
6,5,4,3,2,1, overlapped) ppm. FTꢀIR (KBr): ν 3433 (br, N–H), 2956–2858 (s, C–H), 1705 (C=O
amide I), 1472 (s, C–H), 1390–1361 (m, C–H tertꢀbutyl), 1251 (s, Si–CH3), 1155–1047 (br s, C–
O), 837–777 (s, S–CH3) cmꢀ1. N,Nꢀdiethylꢀ3,6ꢀOꢀdiTBDMS chitosan (6b): Yield: 93%. H
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NMR (400 MHz, CDCl3): δ 0.05 [SiC(CH3)3], 0.87 [Si(CH3)2], 1.45 (Hꢀ8, 8'), 2.38–2.51 (Hꢀ7,
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7'), 3.6–4.0 (Hꢀ6,5,4,3,2,1, overlapped) ppm. FTꢀIR (KBr): ν 3448 (br, N–H), 2957–2858 (s, C–
H), 1705 (C=O amide I), 1472 (s, C–H), 1390–1361 (m, C–H tertꢀbutyl), 1254 (s, Si–CH3),
1105–1066 (br s, C–O), 837–777 (s, Si–CH3) cmꢀ1. N,Nꢀdibutylꢀ3,6ꢀOꢀdiTBDMS chitosan
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(6c): Yield: 89%. H NMR (400 MHz, CDCl3): δ 0.01 [SiC(CH3)3], 0.82 [Si(CH3)2, Hꢀ10, Hꢀ
10'], 1.19 (Hꢀ9, Hꢀ9'), 1.32 (Hꢀ8, Hꢀ8'), 2.57 (Hꢀ7, Hꢀ7'), 3.1–5.0 (Hꢀ6,5,4,3,2,1, overlapped)
ppm. FTꢀIR (KBr): ν 3450 (br, N–H), 2956–2858 (s, C–H), 1705 (C=O amide I), 1471 (s, C–H),
1390–1361 (m, C–H tertꢀbutyl), 1253 (s, Si–CH3), 1105–1071 (br s, C–O), 837–777 (s, Si–CH3)
cmꢀ1. N,Nꢀdihexylꢀ3,6ꢀOꢀdiTBDMS chitosan (6d): Yield: 93%. 1H NMR (400 MHz, CDCl3): δ
0.07 [SiC(CH3)3], 0.89 [Si(CH3)2, Hꢀ12, Hꢀ12'], 1.28–1.41 (Hꢀ11,10,9,8, Hꢀ11',10',9',8'), 2.62
(Hꢀ7, Hꢀ7'), 3.2–5.0 (Hꢀ6,5,4,3,2,1, overlapped) ppm. FTꢀIR (KBr): ν 3433 (br, N–H), 2956–
2857 (s, CꢀH), 1705 (C=O amide I), 1471 (s, C–H), 1390–1361 (m, C–H tertꢀbutyl), 1253 (s, Si–
CH3), 1105–1073 (br s, C–O), 837–777 (s, Si–CH3) cmꢀ1.
2.3.5. Synthesis of N,Nꢀdialkyl chitosan derivatives (7a–d): General procedure. The
corresponding N,Nꢀdialkylꢀ3,6ꢀOꢀdiTBDMS chitosan of 6a–d (2 mmol) was deprotected by
treatment with 1M TBAF solution in NMP (12 mL) at 50 °C for 48 h. The resulting solution was
then dialyzed against deionized water for 2 days, ionꢀexchanged by stirring in 10% NaCl (w/v)
overnight, followed by dialysis against deionized water for 2 days. The resulting compound was
then freezeꢀdried for 2 days, giving the N,Nꢀdialkyl chitosan derivative as an offꢀwhite solid.
N,Nꢀdimethyl chitosan (7a): Yield: 72%. 1H NMR (400 MHz, D2O): δ 3.08 (N–CH3), 3.41 (Hꢀ
2), 3.78–3.86 (Hꢀ6), 3.97 (Hꢀ5), 4.13 (Hꢀ4), 4.25 (Hꢀ3), 5.13 (Hꢀ1) ppm. FTꢀIR (KBr): ν 3442
(O–H), 2956–2881 (s, C–H), 1658 (C=O amide I), 1459 (s, C–H), 1370 (C–H), 1242 (C–N),
1062 (br s, C–O) cmꢀ1. N,Nꢀdiethyl chitosan (7b) : Yield: 61%. H NMR (400 MHz, D2O): δ
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1.35 (Hꢀ8, Hꢀ8'), 3.18–3.40 (Hꢀ2, Hꢀ7, Hꢀ7'), 3.55–4.07 (Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6), 5.06 (Hꢀ1) ppm.
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FTꢀIR (KBr): ν 3433 (O–H), 2930–2881 (s, C–H), 1640 (C=O amide I), 1453 (s, C–H), 1371
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(C–H), 1240 (C–N), 1043 (br s, C–O) cmꢀ1. N,Nꢀdibutyl chitosan (7c): Yield: 58%. H NMR
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(400 MHz, D2O/DCl): δ 0.96 (Hꢀ10, Hꢀ10'), 1.42 (Hꢀ9, Hꢀ9'), 1.81 (Hꢀ8, Hꢀ8'), 3.24–3.59 (Hꢀ7,
Hꢀ7', Hꢀ2), 3.77–3.84 (Hꢀ6), 3.97 (Hꢀ5), 4.12 (Hꢀ4), 4.23 (Hꢀ3), 5.19 (Hꢀ1) ppm. FTꢀIR (KBr): ν
3430 (O–H), 2957–2871 (s, C–H), 1632 (C=O amide I), 1464 (s, C–H), 1376 (C–H), 1248 (C–
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N), 1066 (br s, C–O) cmꢀ1. N,Nꢀdihexyl chitosan (7d): Yield: 65%. H NMR (400 MHz,
CDCl3): δ 0.95 (Hꢀ12, Hꢀ12'), 1.34 (Hꢀ9,10,11, Hꢀ9',10',11'), 1.50 (Hꢀ8, Hꢀ8'), 2.72 (Hꢀ2, Hꢀ7, Hꢀ
7'), 3.49–3.90 (Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6), 4.54 (Hꢀ1) ppm. FTꢀIR (KBr): ν 3431 (O–H), 2955–2858 (s,
C–H), 1631 (C=O amide I), 1466 (s, C–H), 1377 (C–H), 1234 (C–N), 1065 (br s, C–O) cmꢀ1.
2.3.6. Synthesis of N,NꢀdialkylꢀNꢀmethyl chitosan derivatives (8b–d): General procedure.
A mixture of N,Nꢀdialkyl chitosan (1.18 mmol) and NaOH (0.095 g, 2.36 mmol) in NMP (2.5
mL) was stirred for 1 h at room temperature. Methyl iodide (0.29 mL, 4.72 mmol) was then
added dropwise whilst cooling and then the resulting mixture was heated at 50 °C for 24 h. The
addition of the reagents was repeated and continued for another 24 h. After cooling to room
temperature, the reaction mixture was dialyzed against water for 2 days, ionꢀexchanged against
10% NaCl solution, again dialyzed against deionized water for another 2 days, and finally freezeꢀ
dried to obtain 8b–d (scheme 1) as lightꢀbrown “fluffy” material. N,NꢀdiethylꢀNꢀmethyl
1
chitosan (8b): H NMR (400 MHz, D2O): δ 1.34 (Hꢀ8, Hꢀ8'), 2.08 (NCOCH3), 2.88–3.09 (Hꢀ7,
Hꢀ7'), 3.39 (N–CH3), 3.43 (Hꢀ2), 3.57–3.96 (Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6), 5.05–5.08 (Hꢀ1) ppm. FTꢀIR
(KBr): ν 3423 (O–H), 2930–2896 (s, C–H), 1638 (C=O amide I), 1454 (s, C–H), 1372 (C–H),
1240 (C–N), 1058 (br s, C–O) cmꢀ1. N,NꢀdibutylꢀNꢀmethyl chitosan (8c): 1H NMR (400 MHz,
D2O): δ 0.95 (Hꢀ10, Hꢀ10'), 1.41 (Hꢀ9, Hꢀ9'), 1.80 (Hꢀ8, Hꢀ8'), 2.08 (NCOCH3), 3.34 (Hꢀ7, Hꢀ7'),
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3.44 (N–CH3), 3.56 (Hꢀ2), 3.79–4.43 (Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6), 5.16–5.22 (Hꢀ1) ppm. FTꢀIR (KBr):
ν 3416 (O–H), 2959–2874 (s, C–H), 1640 (C=O amide I), 1485 (s, C–H), 1378 (C–H), 1206 (C–
N), 1085 (br s, C–O) cmꢀ1. N,NꢀdihexylꢀNꢀmethyl chitosan (8d): 1H NMR (400 MHz, DMSOꢀ
d6): δ 0.86 (Hꢀ12, Hꢀ12'), 1.28 (Hꢀ9,10,11, Hꢀ9',10',11'), 1.70–1.85 (Hꢀ8, Hꢀ8'), 2.08 (NCOCH3),
2.82–3.03 (Hꢀ7, Hꢀ7'), 3.16 (N–CH3), 3.41–4.39 (Hꢀ2, Hꢀ3, Hꢀ4, Hꢀ5, Hꢀ6), 5.30–5.44 (Hꢀ1)
ppm. FTꢀIR (KBr): ν 3423 (O–H), 2956–2858 (s, C–H), 1640 (C=O amide I), 1466 (s, C–H),
1377 (C–H), 1207 (C–N), 1062 (br s, C–O) cmꢀ1.
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2.3.7. Synthesis of N,Nꢀdibutyl chitosan derivative from native chitosan. Chitosan polymer
(0.5 g, 3.0 mmol) was stirred in acetic acid (10 mL) and butyraldehyde (1.35 mL, 15.3 mmol) at
40 °C for 24 h. The reaction mixture was then cooled down and sodium borohydride (0.58 mg,
15.3 mmol) was added slowly in small portions, and stirring was then continued for 24 h. The
addition of butyraldehyde (15 mmol) and sodium borohydride (15 mmol) was repeated once
more in a similar manner. After 96 h, the reaction mixture was dialyzed against deionized water
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for 2 days and freeze dried to get the product as an offꢀwhite solid. Yield: 76%. H NMR (400
MHz, D2O/DCl): δ 0.96 (Hꢀ10, Hꢀ10'), 1.42 (Hꢀ9, Hꢀ9'), 1.81 (Hꢀ8, Hꢀ8'), 3.24–3.59 (Hꢀ7, Hꢀ7',
Hꢀ2), 3.77–3.84 (Hꢀ6), 3.97 (Hꢀ5), 4.12 (Hꢀ4), 4.23 (Hꢀ3), 5.19 (Hꢀ1) ppm. FTꢀIR (KBr): ν 3430
(O–H), 2957–2871 (s, C–H), 1632 (C=O amide I), 1464 (s, C–H), 1376 (C–H), 1248 (C–N),
1066 (br s, C–O) cmꢀ1.
2.3.8. Synthesis of melittin and LLꢀ37. Synthesis of the two peptides was carried out on a
Syro II (MultiSynTech GmbH) on a TentaGel S Rink Amide 0.24 mmol/g resin (Rapp Polymere
GmbH) using amino acids (5.2 equiv), HOAt (1ꢀhydroxyꢀ7ꢀazabenzotriazole; 5.2 equiv) and
HBTU (2ꢀ(1Hꢀbenzotriazoleꢀ1ꢀyl)ꢀ1,1,3,3ꢀtetramethyluronium hexafluorophosphate; 4.9 equiv)
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in N,Nꢀdimethylformamide (DMF) and N,Nꢀdiisopropylethylamine (DIEA; 9.75 equiv) in Nꢀ
methylpyrrolidone (NMP). Double couplings were used for the syntheses with a coupling time of
2 h each. For Fmoc removal, piperidineꢀDMF (2:3) was used for 3 minutes, followed by 2×15
min deprotection with piperidineꢀDMF (1:4). In between couplings and deprotections, the resin
was washed 3 times with NMP, 2 times with dichloromethane and then 3 times with NMP again.
All the peptides were cleaved by treatment with a mixture of trifluoroacetic acid (TFA),
triethylsilane (TES) and H2O (95:2:3) for 2 h. The TFA solutions were concentrated by nitrogen
flow and the compounds were precipitated with diethylether to yield the crude products. The
peptides were purified by RPꢀHPLC (on a Dionex Ultimate 3000 system) on a preparative C18
column (FeF Chemicals, 200 Å 10 µm C18 particles, 2.1×200 mm) using the following solvent
system: solvent A, water containing 0.1% TFA; solvent B, acetonitrile containing 0.1% TFA.
Gradient elution (0–5 min: 5% to 40% 5–32 min) was applied at a flow rate of 10 mL/min.
Peptide purity was assessed by LCMS on a Dionex Ultimate 3000 system with identification by
ESIꢀMS (MSQ Plus Mass Spectrometer, Thermo).
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Melittin: MS (ESI): calcd for C131H229N39O31[M+H]+: 2846.52 Da; found: m/z 1424.8
[M+2H]2+, 949.8 [M+3H]3+, and 712.5 [M+4H]4+.
LLꢀ37: MS (ESI): calcd for C205H341N61O52[M+H]+: 4492.35 Da; found: m/z: 1123.8
[M+4H]4+, 899.2 [M+5H]5+, 749.7 [M+6H]6+, 642.7 [M+7H]7+.
2.4. Molecular weight determination and solubility tests. Molecular weight (Mw)
determination was carried out using gel permeation chromatography (GPC). GPC measurements
were done using the Polymer Standards Service (PSS) (GmbH, Mainz, Germany), Dionex
Ultimate 3000 HPLC system (Thermo ScientificꢀDionex Softron GmbH, Germering, Germany),
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Dionex Ultimate 3000 HPLC pump and Dionex Ultimate 3000 autosampler (Thermo Scientificꢀ
Dionex Softron GmbH, Germering, Germany), Shodex RIꢀ101 refractive index detector
(Shodex/Showa Denko Europe GmbH, Munich, Germany) and PSS’s ETAꢀ2010 viscometer.
WINGPC Unity 7.4 software (PSS GmbH, Mainz, Germany) was used for data collection and
processing. A series of three columns [Novema 10 µ guard (50 x 8 mm), Novema 10 µ 30 Å
(150 x 8 mm) and Novema 10 µ 1000 Å (300 x 8 mm)] (PSS GmbH, Mainz, Germany) were
used in the HPLC system. Poly(2ꢀvinylpyridine) standards with Mp (1310–256000 Da) and
Dextran (Mw = 62400 Da), from PSS (GmbH, Mainz, Germany) were used for calibration. The
eluent used was 0.1 M NaCl/0.1% TFA solution. Each sample was dissolved in the eluent for 2ꢀ
5 min prior to measurement at a concentration of 1 mg/mL, filtered through a 0.45 ꢁL filter
(Spartan 13/ 0.45 RC, Whatman) before measurement at 25 °C using a flow rate of 1 mL/min.
Each sample had an injection volume of 100 µL and a retention time of 25 min and all the
measurements were done in triplicates.
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The solubility tests of the chitosan derivatives were performed by stirring the compounds in
the solvent for 5 min–24 h at a concentration of 10 mg/mL, which was followed by visual
observation. Samples that were not soluble were diluted to a concentration of 2.5 mg/mL. The
solubility was defined as: fully dissolved in 10% (w/v) concentration (+++), fully dissolved in 2.5
% (w/v) concentration (++), swollen (+/ꢀ), and insoluble (ꢀ).
2.5. Antibacterial tests. The antibacterial tests were assayed, according to standard CLSI
methods for antimicrobial dilution susceptibility tests25 to measure the minimum inhibitory
concentration (MIC) and minimum lethal concentration (MLC)26. The antibacterial activity was
tested against four different bacterial species, that is, two Gram positive bacteria Staphylococcus
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aureus (S.aureus, ATCC 29213) and Enterococcus faecalis (E.faecalis, ATCC 29212), and two
Gram negative bacteria Escherichia coli (E.coli, ATCC 25922) and Pseudomonas aeruginosa
(P.aeruginosa, ATCC 27853), representing clinically important species and strains. The
selection also represents one strain which is more susceptible (S.aureus and E.coli) and one
strain which is more resistant (E.faecalis and P.aeruginosa) in each of the Gram positive and
Gram negative group of bacteria. The broth microdilution method was used to determine the
MIC values using Mueller–Hinton broth at pH 7.2. Blood agar was used to measure the MLC.
Samples were prepared in sterile water at an initial concentration of 4096 µg/mL, which was
then serially diluted by twofold dilutions in a 96ꢀwell plate using Muller–Hinton broth. This gave
a final concentrations varying from 2048 to 1 µg/mL. Gentamicin was used as the positive
control during the test. A standard 0.5 McFarland suspension (1–2 x 108 CFU/mL) was prepared
by direct colony suspension in Mueller–Hinton broth. This suspension was further diluted to
achieve a final test concentration of 5 x 105 CFU/mL in the wells of the microtiter plate. The
microtiter plates were then incubated at 35 °C for 18 h under moistened conditions. The MIC
values were determined as the lowest concentrations of the antibacterial agent to completely
inhibit the visible growth of the microorganism in the microtiter plate. For MLC measurements,
10 µL x 2 (of each of the dilutions that showed no visible growth) was placed on a blood agar
plate and incubated at 35 °C for 18 h. The MLC was determined as the lowest concentration that
achieved a 99.9 % decrease in the viable cells.
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2.6. Hemolysis assay. Hemolysis assays were performed according to previously published
procedures.26 Human red blood cell (RBC) concentrate (6.45 x 1012RBCs/L, total hemoglobin of
201 g/L, 0.15 x 109WBCs/L) was used for testing the hemolytic activity of the chitosan
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derivatives. RBCs (100 µL) were suspended in trisꢀbuffered saline (TBS; 10 mL; pH = 7.2). The
polymer solutions were prepared in TBS at an initial concentration of 4096 µg/mL, and serially
diluted twofold in a 96ꢀwell plate, so as to have a minimum concentration of 1 µg/mL. The RBC
suspension (100 µL) was added to the polymer solutions (100 µL) and incubated at 37 °C with
light shaking for 30 min. Cells treated with TBS and 2% TRITONꢀX were used as negative and
positive controls, respectively. The cell suspensions were centrifuged at 1500 rpm for 10 min,
and the supernatant was used for measuring the absorbance of the released hemoglobin at 540
nm with the Thermo Scientific Multiscan Spectrum. The percentage hemolysis was calculated
using the Equation (4):
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ꢔ − ꢔꢕ
( )
Hemolysis rate % =
( )
4
x 100%
(
)
ꢔꢒꢕꢕ − ꢔꢕ
where A is the absorbance of the polymer solutions, A0 is the absorbance of the negative
control, and A100 is the absorbance of the positive control.
2.7. Cytotoxicity. The cytotoxicity of the chitosan derivatives and peptides melittin and LLꢀ37
was evaluated by XTT assay using the human epithelial colorectal adenocarcinomaꢀderived cell
line, Cacoꢀ2 (ATCC) and by MTT assay using the immortalized human bronchial epithelial cell
line VA10 by previously reported procedures.26,27 Details of the experiments are provided in the
supporting information.
3. RESULTS AND DISCUSSION
3.1. Synthesis. 3.1.1. Nꢀmono and N,Nꢀdialkylation of 3,6ꢀOꢀdiTBDMS chitosan.
Alkylation by reductive amination of chitosan has been accomplished in several studies.13,16,28
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The conventional method usually involves the treatment of chitosan with the aldehyde under
acidic conditions to form the imine, followed by reduction with a suitable reducing agent.
Previous studies have shown that uniform and high DS of alkylation could be achieved in the
case of monoꢀNꢀalkyl chitosan derivatives when 3,6ꢀOꢀdiTBDMS chitosan was used as the
precursor.22 On the other hand, the conventional procedure for reductive monoꢀ and dialkylation,
based on reductive alkylation in acidic aqueous medium, results in a decrease in the DS as the
alkyl chain length increases from methyl onwards.13,29,30 In the current study, monoalkyl chitosan
derivatives with ethyl, butyl, and hexyl chains were synthesized using this precursor. Initially,
TBDMS chitosan was treated with aldehyde in CH2Cl2 to form the imine, which was isolated (to
prevent overalkylation) and then reduced with Na(OAc)3BH to get monoꢀNꢀalkyl chitosan. This
synthetic approach was, therefore, also utilized for the dialkylation of chitosan with the aim of
obtaining a higher DS, particularly with the longer alkyl chains. TBDMS chitosan was first
treated with the corresponding aldehyde (formaldehyde, acetaldehyde, butyraldehyde, and
hexanal) in DCM in the presence of molecular sieves and a catalytic amount of acetic acid in
order to drive imine formation31,32 to completion. The imine was then subjected to reduction in
situ and in the presence of a reducing agent (NaBH4) to give the partially N,Nꢀdialkylated
product. This product, when treated with similar equivalents of the aldehyde (to form the second
imine) followed by NaBH4, resulted in the formation of the N,Nꢀdialkylated product (scheme 1).
This procedure gave 100% dialkylation, even with the longer alkyl chains. The initial addition of
a large excess of aldehyde and NaBH4 did not result in full dialkylation, as NaBH4 also reduces
the excess aldehyde present in the medium to an alcohol, thereby preventing further
condensation. Therefore, the repeated addition of the reagents is a requirement for the formation
of the fully disubstituted product. As a control, the dialkylation reaction was also performed on
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Scheme 1.Synthetic route for preparation of quaternary chitosan derivatives. Reagents and
conditions: (i) MeI, NMP, 40 ⁰C; (ii) TBAF (1 M), NMP, 50 ⁰C; (iii) aldehyde, TEA, CH2Cl2, 45
⁰C; (iv) Na(OAc)3BH, AcOH, CH2Cl2, room temperature; (v) Me2SO4, LiCO3, CH2Cl2, 50 ⁰C;
(vi) TBAF (1 M), NMP, 50 ⁰C; (vii) aldehyde, NaBH4, CH2Cl2, room temperature, (viii) TBAF
(1 M), NMP, 50 ⁰C; (ix) MeI, NaOH, NMP, 50 ⁰C.
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the unprotected chitosan using the conditions of butyraldehyde/AcOH/NaBH4. This reaction,
when carried out using the same equivalents of both the aldehyde and NaBH4 as with the
TBDMSꢀprotected chitosan reaction, gave only around 65% diꢀsubstitution. This is comparable
to the DS (0.5) of N,Nꢀdibutyl chitosan16 obtained in earlier studies under acidic conditions.
Thus, the use of TBDMSꢀprotected chitosan is found to be necessary in order to obtain full N,Nꢀ
dialkylation with the help of simple reaction conditions in organic solvent. The removal of the
diꢀTBDMS protection groups was carried out by treatment with a 1M solution of TBAF in
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Figure 1.A) 1H NMR spectra and B) FTꢀIR spectra of (a) 3,6ꢀOꢀdiTBDMS chitosan, (b) N,Nꢀ
dibutylꢀ3,6ꢀOꢀdiTBDMS chitosan and (c) N,Nꢀdibutyl chitosan.
NMP.22 The products were then purified by dialysis in order to remove the excess reagents and
side products. In cases where traces of silyl groups were retained, the deprotection procedure was
repeated once more.
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Figure 1A shows a comparison of the H NMR spectra for the N,Nꢀdibutyl reaction. Spectrum
(a) shows TBDMS chitosan, with the silyl peaks appearing at 0.05 and 0.89 ppm. After the
dialkylation reaction, the product shows new peaks appearing at 1.19 (Hꢀ9, Hꢀ9'), 1.32 (Hꢀ8, Hꢀ
8'), and 2.57 (Hꢀ7, Hꢀ7') ppm in addition to the silyl peaks. This confirms the attachment of the
butyl group to the polymer chain. Also, the absence of any imine protons at 7.4 ppm23 indicates
the complete reduction of the imine intermediate. However, owing to the broad nature of the
peaks, it is difficult to ascertain the DS of the protected compound. After deprotection of the silyl
groups, the peaks become well resolved, as seen in spectrum (c), and the exact DS could be
determined from the proton integrals. Also, each of the prochiral Hꢀ7 and Hꢀ7' protons in the
butyl chains, which were initially merging with the Hꢀ2 proton in spectrum (b), can be seen to be
split into two peaks for each proton. They appear at 3.25, 3.39, 3.51, and 3.59 ppm (merging
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Figure 2.1H NMR spectra of (i) N,Nꢀdihexyl chitosan, (ii) N,Nꢀdiethyl chitosan, (iii) N,Nꢀdibutyl
chitosan, and (iv) N,Nꢀdimethyl chitosan.
with the Hꢀ2 peak) after deprotection. The strong correlation of Hꢀ7 protons with Hꢀ2 as well as
the correlation between Hꢀ8, Hꢀ9, and Hꢀ10 protons in the COSY spectrum (not shown) also
confirms the dibutylation of chitosan.
Figure 1B shows a comparison of FTꢀIR spectra for the same reaction. Spectra (a) and (b)
show stretching frequencies at 1361–1390 (m, C–H tertꢀbutyl), 1255 (s, Si–CH3), and 778–837
(s, Si–CH3) cmꢀ1, corresponding to the TBDMS group. The enhanced C–H stretching intensity in
the 2871–2957 cmꢀ1 region, accompanied by additional peaks at 1376 (C–H) and 1248 (C–N) cmꢀ
1
in spectra (b) and (c), indicates the presence of the butyl group. The broad O–H band at 3430
cmꢀ1 can be observed after the removal of the silyl groups in spectrum (c).
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An overlay of the H NMR spectra for the four dialkylated compounds, that is, N,Nꢀdimethyl,
N,Nꢀdiethyl, N,Nꢀdibutyl, and N,Nꢀdihexyl chitosan, is shown in Figure 2. Each spectrum shows
distinct peaks for the corresponding alkyl groups, that is, 3.08 ppm for the N(CH3)2 group in (iv)
and the absence of any peak at 3.0 ppm confirms the absence of monoꢀNꢀmethylation;22 1.35 and
3.18–3.40 ppm correspond to the –CH3 and –CH2– protons, respectively, for the N,Nꢀdiethyl
groups in (iii); 0.96 (–CH3), 1.42 (–CH2–), 1.80 (–CH2–) and 3.26–3.59 ppm (N–CH2–)
correspond to the dibutyl groups; 0.97 (–CH3), 1.35 [–(CH2)–], 1.49–1.59 [–(CH2)–], and 2.72
ppm (N–CH2–) correspond to the dihexyl groups. The NMR spectra for all of the derivatives
were measured in D2O, except for the N,Nꢀdihexyl chitosan, which was insoluble in water;
hence, the NMR spectrum was taken in CDCl3. The DS of each of the N,Nꢀdialkyl derivative was
calculated using equation (2), and the dialkylation was found to be 100% in each case. The
presence of a sharp single Hꢀ1 peak in all spectra also indicates full dialkylation and the absence
of any residual monoꢀalkylation.
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3.1.2. Quaternization of the Nꢀmono and N,Nꢀdialkyl chitosan derivatives. The Nꢀalkyl
chitosan derivatives were quaternized by using methylating reagents. The monoꢀNꢀalkyl
derivatives were quaternized by reacting them with Me2SO4 in CH2Cl2,22 and this resulted in a
high degree of quaternization (DQ). The quaternary derivatives were then deprotected using
TBAF/NMP. The DQ values were calculated using equations previously reported.22 The degree
of dimethylation ranged from 62 to 74%, which is similar to the range (65–72%) obtained
previously.22 The N,N,Nꢀtrimethyl chitosan homopolymer was also synthesized from the
TBDMSꢀprotected chitosan using previous procedures.22 However, no product conversion was
observed when these conditions were applied for the methylation of the N,Nꢀdialkyl derivatives.
Various conditions were investigated in order to optimize the quaternization reaction (Table 1).
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Table 1.Investigated reaction conditions for the dialkylation and the quaternization reactions
performed on N,Nꢀdibutyl chitosan derivatives.
7
8
9
Reaction/ starting material
Reagents
Temperature Time
(°C)
DS
(%)
(h)
96
96
48
48
48
48
48
48
48
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Dialkylation/ Chitosan
Butyraldehyde/NaBH4/AcOH
Butyraldehyde/NaBH4/DCM
DCM/Me2SO4
40
65
100
0
Dialkylation/ 3,6ꢀOꢀdiTBDMS chitosan
Quaternization/ N,NꢀdibutylꢀdiTBDMS chitosan
RT
50
NMP/MeI/NaOH
DCM/CF3SO3Me
NMP/MeI/Cs2CO3
DMF/H2O/MeI
50
0
RT
50
30
0
Quaternization/ N,Nꢀdibutyl chitosan
RT
40
0
NMP/ MeI
20
75
NMP/MeI /NaOH*
40
*This reaction was done twice.
Methylation of the TBDMSꢀprotected dibutyl derivative was proven to be too difficult and some
DQ (ca. 30%) was only possible when using the highly reactive methyltriflate. Although full
quaternization could be obtained with the dimethyl compound, only limited methyl substitution
could be seen in the case of diethyl and dibutyl, and no methylation of N,Nꢀdihexylꢀ3,6ꢀOꢀ
diTBDMS chitosan could be observed (data not shown). In contrast, a high degree of methyl
substitution was observed in the case of monoꢀNꢀalkyl chains. These results showed that the
amino N atom in diꢀTBDMS chitosan is shielded from the reaction by steric hindrance after the
second alkyl has been introduced. The incoming electrophilic methylating reagent does not
appear to have sufficient access to the free electron pair on the N atom, as required for reaction.
The bulky TBDMS protection groups will also contribute to this hindrance. The methylation
reaction was, therefore, attempted on the deprotected dialkylated chitosan derivative after the
bulky TBDMS protection groups had been removed. As seen in Table 1, previously established
methylation procedures like MeI/NaOH/DMFꢀH2O33 and DMS/NaOH/NaCl/H2O34 did not result
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in quaternization. In contrast, quaternization of around 20% was obtained when using MeI in
NMP, and this could be further improved by using NaOH as a base. This reaction was repeated
twice in order to obtain a methyl substitution as high as 75%. This result showed considerable
improvement compared to the previously reported synthesis of N,NꢀdibutylꢀNꢀmethyl chitosan,16
in which the degree of N,Nꢀdialkylation was reported to be 0.5 and the DQ was 0.28. Hence, a
high DQ with longer alkyl chains could only be obtained after removal of the bulky TBDMS
groups. The DQ values were calculated using equation (3), as shown in Table 2. The similarity in
the DQ values obtained from the N,Nꢀdialkyl derivatives with those of the monoꢀNꢀalkyl
derivatives gave us a series of uniformly quaternized and highly waterꢀsoluble chitosan
derivatives, which were suitable for antimicrobial testing.
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The H NMR spectrum of N,NꢀdibutylꢀNꢀmethyl chitosan shows a singlet appearing at 3.44
ppm clearly showing the Nꢀmethyl peak. The quaternization is also evident by the splitting of the
Hꢀ1 proton and broadening of the dibutyl peak at 1.82 ppm in comparison to that of the N,Nꢀ
dibutyl chitosan derivative (figure S17, supporting information) . Although quaternization of the
1
unprotected chitosan leaves the possibility of some Oꢀmethylation during the reaction, the H
NMR spectra did not show any prominent Oꢀmethyl peak (3.35 ppm)33 indicating Nꢀmethylation
to be the primary reaction.
3.2. Molecular weight and solubility properties. The Mw of the starting chitosan material and
the final products was determined using size exclusion chromatography. No change in Mw
values was observed during the analysis of each sample. The results showed that the synthetic
modification was accompanied by significant degradation of the polymer backbone. The Mw
analysis of some intermediates showed that this mainly occurred during the preparation of the
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Table 2.Physicochemical properties of quaternary chitosan derivatives
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6
7
8
9
Compound
Degree of Quaternisation
(DQ%)
Isolated
Yield (%)
Mw
(Da)
MV(Da)
[viscosity
average Mw]
PDI
Solubility
Aqueous
DMSO
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30
31
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N,N,Nꢀtrimethyl
chitosan (3)
100
66
74
62
72
75
61
65
45
43
52
45
42
48
15730
9924
8725
8766
7875
9248
11774
15725
9919
1.42
1.03
1.02
1.05
1.05
1.01
1.23
+++
+++
+++
+++
+++
+++
+++
–
NꢀethylꢀN,Nꢀ
dimethylchitosan (5a)
–
NꢀbutylꢀN,Nꢀ
dimethylchitosan (5b)
8721
+/–
++
NꢀhexylꢀN,Nꢀ
dimethylchitosan (5c)
8762
N,NꢀdiethylꢀNꢀ
methylchitosan (8b)
7871
–
N,NꢀdibutylꢀNꢀ
methylchitosan (8c)
9245
+/–
+++
N,NꢀdihexylꢀNꢀ
methylchitosan (8d)
11770
PDI = polydispersity index; +++ = fully dissolved in 10% (w/v) concentration; ++ = fully
dissolved in 2.5% (w/v) concentration; +/– = swollen and – = insoluble; MV was determined by
GPC.
mesylate salt, which required that the chitosan polymer was dissolved in methane sulfonic
acid. The Mw for chitosan sample which was initially 294 kDa, was reduced to 30 kDa after this
step and the conversion to the protected precursor. It has previously been observed that the
protection and deprotection of the chitosan polymer also leads to degradation of the backbone.35
In another study, it was found that arylation followed by methylation at the amino group of
chitosan was accompanied by a significant Mw reduction.36 Hence, Mw analysis of the final
compounds is essential, as the Mw can vary significantly from that of the starting material.37 The
final quaternary chitosan derivatives had similar Mw values, ranging from 7 to 15 kDa, as shown
in Table 2. The polydispersity index (PDI) of the derivatives ranged from 1.01 to 1.42, whereas
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the PDI for the starting material was 2.3. This uniformity and lowering in the Mw and PDI values
obtained after synthetic modification has also been observed in our previous studies.37,38
The hydrolysis of the glycosidic bonds in the longer polymer chains is higher and this leads to
low Mw fractions, which after purification using dialysis membrane (Mw cutꢀoff 3000 Da) gives
compounds having lower and uniform PDI values.
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3.3. Antibacterial properties of the Nꢀquaternary chitosan derivatives. In order to have an
overview of the effect that the quaternary polymers would exert on bacteria, the series of
quaternary chitosan derivatives were tested for activity against two Gram positive (S.aureus and
E.faecalis) and two Gram negative (E.coli and P.aeruginosa) bacterial species. The activity of
the polymers were compared against two quaternary ammonium disinfectants, cetyl pyridinium
chloride (CPC) and benzalkonium chloride (BAC), and two antimicrobial peptides, melittin and
LLꢀ37, which are also polycationic. CPC is an antiseptic used in oral rinses and CPC formulation
studies have shown bacterial detachment in vitro and bactericidal effects in vivo.39
Benzalkonium chloride is a cationic surfaceꢀacting reagent that is mostly used to disinfect
medical equipment and is also used in the food industry.40 These two commonly used
disinfectants were, therefore, included in our antibacterial studies. Antimicrobial peptides
(AMPs) are natural compounds that have a broad spectrum of activity against bacteria and are
known to kill the bacterial cell through interactions with the cell membrane. As chitosan is also
known to mostly interact with bacteria at the cell surface, a comparative study of the activity of
these natural biocides with that of the chitosan derivatives was performed. Two AMPs, melittin
(present in bee venom) and the human cathelicidin LLꢀ37, were synthesized and tested against
the four different bacterial strains. The activity of melittin was found to be consistent with
previous studies.41 LLꢀ37 also showed comparable activity to earlier studies against three
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strains,42 while its activity against S.aureus was found to be comparatively lower, probably due
to the degradation of this peptide caused by S.aureus derived proteinases43. As shown in Table 3,
all of the chitosan derivatives were active against the four strains within the range of
concentrations (1–2048 µg/mL) measured. Compounds 3, 5a, and 5b showed higher activity
(with low MIC) against S.aureus while all the other derivatives displayed a descending order in
activity with increasing hydrophobicity. The most active compound N,NꢀdiethylꢀNꢀmethyl
chitosan (5a) having a MIC of 4 µg/mL, was found to be similar in activity as melittin (MIC = 4
µg/mL) and considerably higher in activity (7ꢀdilutions) than LLꢀ37 (MIC = 512 µg/mL). On the
other hand, this compound was less active than CPC (MIC ≤ 0.5) and BAC (MIC ≤ 0.5) against
S.aureus. A different behavior in activity was observed against the other Gram positive bacteria,
E.faecalis. The activity was seen to be proportional to the length of the alkyl chain, as it
increased from methyl to hexyl in both series. A similar trend in activity was also seen against
the Gram negative bacteria E.coli. This result was consistent with the previous study, where the
quaternary chitosan derivative with a longer chain length was found to be more active than that
with a shorter chain against E.coli.44 Thus, the N,NꢀdihexylꢀNꢀmethyl chitosan (8d) and Nꢀhexylꢀ
N,Nꢀdimethyl chitosan (5c) were the most active compounds against E.faecalis (MIC value 16
and 64 µg/mL, respectively) and E.coli (MIC value 16 µg/mL for both) amongst all of the
derivatives measured. Their MIC values revealed that their activity was comparable to that of
melittin, but was considerably higher (4–6 dilutions) than that of LLꢀ37, especially against
E.faecalis, that was chosen to represent relatively resistant Gram positive strain. The two
derivatives also remained equally active to CPC and BAC against E.coli, but showed less
efficacy against E.faecalis. Overall, the increase in activity with chain length was clearly
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Table 3.Biological activity data for the chitosan derivatives, as well as for benzalkonium
5
6
chloride, cetyl pyridinium chloride, melittin, and LLꢀ37.
7
8
9
Gram positive bacteria
Gram negative bacteria
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S.aureus
E.faecalis
E.coli
P.aeruginosa
Compound
Hemolytic
activity
HC50 (µg/mL)
MIC(µg/mL)
(Selectivity
HC50/MIC)
MIC(µg/mL)
(Selectivity
HC50 /MIC)
MIC(µg/mL)
(Selectivity
HC50 /MIC)
MIC(µg/mL)
(Selectivity
HC50 /MIC)
32
128
64
512
˃1024
(˃32)
(˃8)
(˃16)
(˃2)
(3)
4
128
(˃8)
64
512
˃1024
36
(˃256)
(˃16)
(˃2)
(5a)
32
64
32
512
(1.12)
(0.56)
(1.12)
(0.07)
(5b)
16
256
16
16
32
(0.06)
(1)
(1)
(0.5)
(5c)
(8b)
384
20
256
512
512
1024
(1.5)
(0.75)
(0.75)
(0.37)
512
128
32
512
(0.04)
(0.15)
(0.62)
(0.04)
(8c)
40
1024
64
(0.62)
16
1024
(0.04)
(0.04)
(2.5)
(8d)
Benzalkonium chloride 28
≤0.5
≤0.5
16
64
(≥56)
(≥56)
(1.75)
(0.43)
Cetyl pyridinium
chloride
≤0.5
≤0.5
(~1)
≤0.5
(~1)
16
256
(<0.05)
(<0.05)
Melittin
8*
4
(2)
8
(1)
16
(0.5)
64
(0.12)
LLꢀ37
336**
512
(0.65)
1024
(0.32)
32
(10.5)
128
(2.6)
The HC50 values for Melittin and LLꢀ37 were obtained from literature (*45,46, **47) due to
insufficient material for testing hemolytic activity. The MLC values for all the compounds were
also determined and these are provided in the supporting information.
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observed against these two bacterial strains, although, the Nꢀmonoalkyl quaternary compounds
displayed better inhibition than the corresponding N,Nꢀdialkyl derivatives. On the other hand, the
Gram negative P.aeruginosa, chosen to represent relatively resistant Gram negative strain, was
equally inhibited by all the quaternary derivatives, although at higher concentrations in most
instances. No ascending or descending trend in activity could be observed with increasing chain
length or hydrophobicity. Most of the derivatives displayed an MIC value within 512–1024
µg/mL, except for 5c, which showed high activity (MIC = 32 µg/mL) within the monoꢀalkyl
series, as seen in table 3. However, this increase in activity was not observed in the case of the
dialkyl series. The lowering in activity against P.aeruginosa could also be seen for CPC (256
µg/mL), BAC (64 µg/mL), melittin (64 µg/mL), as well as LLꢀ37 (128 µg/mL). In a previous
study, it was reported that an increase in the quaternization and the length of the alkyl chain led
to an increase in antibacterial activity of chitosan derivatives against S.aureus and E.coli.4 In this
current study, the effect of the variation in chain length and hydrophobicity was found to be
dependent on the bacterial strain which can be seen in figure 4 (1 and 2). In order to determine
whether the quaternary derivatives were capable of inducing bactericidal effect, the MLC values
were also determined. As seen (table S2, supporting information), the MLC were in close range
(either identical or 1–2 dilutions higher than the MIC) to the MIC indicating that the derivatives
were bactericidal.
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The activity of unmodified chitosan at pH 7.2 was observed to be in the 1024–2048 µg/mL
range for S.aureus and E.coli48, while these quaternary derivatives exhibited higher antibacterial
properties at neutral pH. The behaviour of the chitosan derivatives towards the four bacterial
species is believed to be related to the difference in the resistance mechanism of each strain as
well as the difference in the cell wall composition. The cell wall of Gram positive S.aureus and
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E.faecalis is comparable, but E.faecalis harbors more of the genes that code for resistance. Such
genes prevent the destruction of the cell wall, the protein production and even the production of
folic acid making this strain less susceptible to antibacterial agents as compared to S.aureus.49 In
case of Gram negative bacteria also, P.aeruginosa is found to be more resistant than E.coli
because of similar reasons.50 The high activity of compound 5a against S.aureus indicated that
the single ethyl group could provide optimum lipophilicity for membrane disruption and
additional hydrophobicity was not necessary. The enhancement of antibacterial efficacy due to
the presence of long chains (in 5c and 8d) could be observed in case of E.faecalis unlike
S.aureus. Many commonly used antimicrobial agents and antibiotics are incapable of killing
E.faecalis. In our study, all the derivatives were bactericidal towards this strain. The Gram
negative E.coli bacteria were also seen to be highly inhibited by the longer alkyl chain
derivatives. This result is in agreement with a previous study, where it was shown that waterꢀ
soluble chitosan derivatives with hydrophobic substituents interact with the lipid A fragment,
which is important for the neutralization of lipopolysaccharide in Gram negative bacteria.51 The
lowest activity shown by all the derivatives towards P.aeruginosa is due to the fact that this
strain is usually found to be highly resistant towards several antibiotics and amino glycosides
owing to the very poor membrane permeability of the outer membrane.52
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The monoꢀNꢀalkylꢀN,Nꢀdimethyl chitosan derivatives were in general, more active than the N,Nꢀ
dialkylꢀNꢀmethyl chitosan derivatives. The variation of activity with chain length against a
particular bacterial strain was found to be similar within the mono alkyl and the dialkyl series as
seen in figure 3. Thus it is apparent that the length of the alkyl chain is the main factor
influencing the activity of the derivatives, hence increasing the hydrophobicity from Nꢀalkyl to
N,Nꢀdialkyl did not enhance their antibacterial properties.
ACS Paragon Plus Environment
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