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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 NAlkyl and N,N-Dialkyl Chitosan Derivatives

Priyanka Sahariaha, Berglind E. Benediktssdóttira, Martha Á. Hjálmarsdóttirb, Olafur E. Sigurjonssonc,d, Kasper K. Sørensene, Mikkel B. Thygesene, Knud J. Jensene, Már Mássona,*s a

Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavik, Iceland

b

Department of Biomedical Science, Faculty of Medicine, University of Iceland, Stapi, Hringbraut 31, 101 Reykjavik, Iceland

c

The REModeL Lab, The Blood Bank, Landspitali University Hospital, Snorrabraut 60, 105 Reykjavik, Iceland

d

Institute of Biomedical and Neural Engineering, Reykjavik University, Menntavegur 1, 101, Reykjavik, Iceland

ACS Paragon Plus Environment

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e

Department of Chemistry, Faculty of Science, Centre for Carbohydrate Recognition and Signalling, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Fredriksberg C, Copenhagen, Denmark

Keywords: 3,6,-O-diTBDMS chitosan; N-alkyl chitosan; Quaternary chitosan; Antibacterial activity; Hemolytic activity; Structure-activity relationship

Abstract

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-tertbutyldimethylsilylchitosan 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 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 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 cetylpyridiniumchloride and benzalkoniumchloride as well as the antimicrobial peptides melittin and LL-37.


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1. INTRODUCTION Chitin represent a class of linear polysaccharides comprised of N-acetyl-D-glucosamine units linked together with β-(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 N-alkyl chitosan have been shown to be effective as antimicrobial agents,4-6 apart from finding application in the field of tissue engineering,7 DNA delivery,8 drug delivery,9 and membrane coating.10 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 N-alkyl or pyridinium groups had enhanced antibacterial activity compared to native chitosan. Surface-quaternized derivatives of chitosan carrying high positive charge density and containing hydrophobic N-substituents like benzyl moieties were shown to inhibit the growth of Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) more strongly than derivatives containing N-propyl 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. 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 N-selective and results in mono- 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 N-alkyl 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 (11–15-fold 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.10,17-19 The possible reason for this is that Nalkylation 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 N-alkyl and N,N-dialkyl 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 TBDMS-protected chitosan (TBDMS = tert-butyldimethylsilyl). 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 Nmodification.20,21 Recently, TBDMS protection has been utilized in reductive amination to yield mono-N-alkylated 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 well-characterized 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 N-alkyl quaternary chitosan derivatives, differing in hydrophobicity (i.e. N-alkyl 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 adenocarcinoma-derived cell line, Caco-2. 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.

2. EXPERIMENTAL 2.1. Materials. Chitosan polymer (S030626-2) was obtained from Genis ehf (Reykjavik, Iceland). Its degree of deacetylation (DA) was found to be 94%, as obtained from the integrals of 1

H 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 Float-A-Lyzers (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).

2.2. Characterization. Detailed characterization of the compounds was performed using 1H and COSY NMR and IR spectroscopy. 1H 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, DMSO-d6, and D2O in concentrations of 10 to 15 mg/mL. The N-acetyl peak (2.08 ppm)24 was used as the internal reference in all spectra. FT-IR measurements were performed with an AVATAR 370 FT-IR 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 N-alkyl 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: ∫

1) Degree of acetylation, DA = ∫ ˣ x 100 (1) 2) DS for N,N-dialkylation, A =

∫ ∫

ˣ x 100 (2)


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∫ !

3) DS for N,N-dialkyl-N-methyl, B = ∫ ,´ ˣ x 100 (3) 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 N-acetyl 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,N-dialkyl-N-methyl and the N,N-dialkyl units respectively. 2.3. Synthesis. 2.3.1. 3,6-O-diTBDMS chitosan (1). 3,6-O-diTBDMS chitosan 1 was synthesized via the chitosan mesylate salt, according to a previously published procedure.21

2.3.2. Synthesis of N,N,N-trimethyl chitosan (3). N,N,N-trimethyl 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 N-methyl-2-pyrrolidone (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 freeze-dried to afford N,N,N-trimethyl3,6-O-diTBDMS 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 ion-exchanged 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 freeze-dried, resulting in light brown and “fluffy” N,N,N-trimethyl chitosan chloride 3. Yield: 74%. 1H NMR (400 MHz, D2O): δ 2.08 (NCOCH3), 3.35 [N(CH3)3], 3.75 (H2), 3.90 (H-6), 3.99 (H-5), 4.36 (H-4), 4.47 (H-3), 5.49 (H-1) ppm. FT-IR (KBr): ν 3386 (O–H), 2930 (s, C–H), 1639 (C=O amide I), 1482 (s, C–H), 1052 (br s, C–O) cm-1.


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2.3.3. Synthesis of N-alkyl-N,N-dimethyl chitosan derivatives (5a–c): General procedure. The N-alkyl-N,N-dimethyl chitosan derivatives were synthesized according to a previously developed procedure.22 Briefly, 3,6-O-diTBDMS 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 air-dried. This material was then re-dissolved 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 N-alkylated 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 freeze-dried for 2 days. The N-alkyl-N,N-dimethyl-diTBDMS 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, ion-exchanged by stirring in 10% NaCl (w/v) overnight, followed by dialysis against deionized water for 2 days. The resulting compounds were then freeze-dried for 2 days, giving the N-alkyl-N,N-dimethyl chitosan derivatives 5a–c (scheme 1) as light yellow solids. N-ethyl-N,N-dimethyl chitosan (5a): 1H NMR (400 MHz, D2O): δ 1.44 (H-8), 2.08 (NCOCH3), 3.25 [N(CH3)2], 3.33 (N–CH3), 3.46–3.79 (H-2, H-7), 3.89–3.96 (H-5, H-6), 4.36 (H-4), 4.50 (H-3), 5.52 (H-1) ppm. FT-IR (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) cm-1. N-butyl-N,N-dimethyl chitosan (5b): 1H NMR (400 MHz, D2O): δ 0.91 (H-10), 1.36 (H-9), 1.86 (H-10), 2.08 (NCOCH3), 3.27 [N(CH3)2], 3.34 (N–CH3), 3.47–3.9 (H-2, H-7), 3.87–4.50 (H-3, H-4, H-5, H-6), 5.30–5.54 (H-1) ppm. FT-IR (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) cm-1. Nhexyl-N,N-dimethyl chitosan (5c): 1H NMR (400 MHz, D2O): δ 0.95 (H-12), 1.40 (H-9, H-10, H-11), 1.87 (H-8), 2.08 (NCOCH3), 3.27 [N(CH3)2], 3.5–3.78 (H-2, H-7), 4.37–4.49 (H-3, H-4, H-5, H-6), 5.25–5.54 (H-1) ppm. FT-IR (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) cm-1.

2.3.4. Synthesis of N,N-dialkyl-3,6-O-diTBDMS 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,Ndialkyl-3,6-O-diTBDMS chitosan. N,N-dimethyl- 3,6-O-diTBDMS chitosan (6a): Yield: 91%. 1

H NMR (400 MHz, CDCl3): δ 0.05 [SiC(CH3)3], 0.87 [Si(CH3)2], 3.3 [N(CH3)2], 3.6–4.0 (H-

6,5,4,3,2,1, overlapped) ppm. FT-IR (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 tert-butyl), 1251 (s, Si–CH3), 1155–1047 (br s, C– O), 837–777 (s, S–CH3) cm-1. N,N-diethyl-3,6-O-diTBDMS chitosan (6b): Yield: 93%. 1H NMR (400 MHz, CDCl3): δ 0.05 [SiC(CH3)3], 0.87 [Si(CH3)2], 1.45 (H-8, 8'), 2.38–2.51 (H-7,


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7'), 3.6–4.0 (H-6,5,4,3,2,1, overlapped) ppm. FT-IR (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 tert-butyl), 1254 (s, Si–CH3), 1105–1066 (br s, C–O), 837–777 (s, Si–CH3) cm-1. N,N-dibutyl-3,6-O-diTBDMS chitosan (6c): Yield: 89%. 1H NMR (400 MHz, CDCl3): δ 0.01 [SiC(CH3)3], 0.82 [Si(CH3)2, H-10, H10'], 1.19 (H-9, H-9'), 1.32 (H-8, H-8'), 2.57 (H-7, H-7'), 3.1–5.0 (H-6,5,4,3,2,1, overlapped) ppm. FT-IR (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 tert-butyl), 1253 (s, Si–CH3), 1105–1071 (br s, C–O), 837–777 (s, Si–CH3) cm-1. N,N-dihexyl-3,6-O-diTBDMS chitosan (6d): Yield: 93%. 1H NMR (400 MHz, CDCl3): δ 0.07 [SiC(CH3)3], 0.89 [Si(CH3)2, H-12, H-12'], 1.28–1.41 (H-11,10,9,8, H-11',10',9',8'), 2.62 (H-7, H-7'), 3.2–5.0 (H-6,5,4,3,2,1, overlapped) ppm. FT-IR (KBr): ν 3433 (br, N–H), 2956– 2857 (s, C-H), 1705 (C=O amide I), 1471 (s, C–H), 1390–1361 (m, C–H tert-butyl), 1253 (s, Si– CH3), 1105–1073 (br s, C–O), 837–777 (s, Si–CH3) cm-1. 2.3.5. Synthesis of N,N-dialkyl chitosan derivatives (7a–d): General procedure. The corresponding N,N-dialkyl-3,6-O-diTBDMS 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, ion-exchanged by stirring in 10% NaCl (w/v) overnight, followed by dialysis against deionized water for 2 days. The resulting compound was then freeze-dried for 2 days, giving the N,N-dialkyl chitosan derivative as an off-white solid. N,N-dimethyl chitosan (7a): Yield: 72%. 1H NMR (400 MHz, D2O): δ 3.08 (N–CH3), 3.41 (H2), 3.78–3.86 (H-6), 3.97 (H-5), 4.13 (H-4), 4.25 (H-3), 5.13 (H-1) ppm. FT-IR (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) cm-1. N,N-diethyl chitosan (7b) : Yield: 61%. 1H NMR (400 MHz, D2O): δ 1.35 (H-8, H-8'), 3.18–3.40 (H-2, H-7, H-7'), 3.55–4.07 (H-3, H-4, H-5, H-6), 5.06 (H-1) ppm.


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FT-IR (KBr): ν 3433 (O–H), 2930–2881 (s, C–H), 1640 (C=O amide I), 1453 (s, C–H), 1371 (C–H), 1240 (C–N), 1043 (br s, C–O) cm-1. N,N-dibutyl chitosan (7c): Yield: 58%. 1H NMR (400 MHz, D2O/DCl): δ 0.96 (H-10, H-10'), 1.42 (H-9, H-9'), 1.81 (H-8, H-8'), 3.24–3.59 (H-7, H-7', H-2), 3.77–3.84 (H-6), 3.97 (H-5), 4.12 (H-4), 4.23 (H-3), 5.19 (H-1) ppm. FT-IR (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) cm-1. N,N-dihexyl chitosan (7d): Yield: 65%. 1H NMR (400 MHz, CDCl3): δ 0.95 (H-12, H-12'), 1.34 (H-9,10,11, H-9',10',11'), 1.50 (H-8, H-8'), 2.72 (H-2, H-7, H7'), 3.49–3.90 (H-3, H-4, H-5, H-6), 4.54 (H-1) ppm. FT-IR (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) cm-1.

2.3.6. Synthesis of N,N-dialkyl-N-methyl chitosan derivatives (8b–d): General procedure. A mixture of N,N-dialkyl 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, ion-exchanged against 10% NaCl solution, again dialyzed against deionized water for another 2 days, and finally freezedried to obtain 8b–d (scheme 1) as light-brown “fluffy” material. N,N-diethyl-N-methyl chitosan (8b): 1H NMR (400 MHz, D2O): δ 1.34 (H-8, H-8'), 2.08 (NCOCH3), 2.88–3.09 (H-7, H-7'), 3.39 (N–CH3), 3.43 (H-2), 3.57–3.96 (H-3, H-4, H-5, H-6), 5.05–5.08 (H-1) ppm. FT-IR (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) cm-1. N,N-dibutyl-N-methyl chitosan (8c): 1H NMR (400 MHz, D2O): δ 0.95 (H-10, H-10'), 1.41 (H-9, H-9'), 1.80 (H-8, H-8'), 2.08 (NCOCH3), 3.34 (H-7, H-7'),


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3.44 (N–CH3), 3.56 (H-2), 3.79–4.43 (H-3, H-4, H-5, H-6), 5.16–5.22 (H-1) ppm. FT-IR (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) cm-1. N,N-dihexyl-N-methyl chitosan (8d): 1H NMR (400 MHz, DMSOd6): δ 0.86 (H-12, H-12'), 1.28 (H-9,10,11, H-9',10',11'), 1.70–1.85 (H-8, H-8'), 2.08 (NCOCH3), 2.82–3.03 (H-7, H-7'), 3.16 (N–CH3), 3.41–4.39 (H-2, H-3, H-4, H-5, H-6), 5.30–5.44 (H-1) ppm. FT-IR (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) cm-1.

2.3.7. Synthesis of N,N-dibutyl 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 for 2 days and freeze dried to get the product as an off-white solid. Yield: 76%. 1H NMR (400 MHz, D2O/DCl): δ 0.96 (H-10, H-10'), 1.42 (H-9, H-9'), 1.81 (H-8, H-8'), 3.24–3.59 (H-7, H-7', H-2), 3.77–3.84 (H-6), 3.97 (H-5), 4.12 (H-4), 4.23 (H-3), 5.19 (H-1) ppm. FT-IR (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) cm-1. 2.3.8. Synthesis of melittin and LL-37. 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 (1-hydroxy-7-azabenzotriazole; 5.2 equiv) and HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; 4.9 equiv)


12

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in N,N-dimethylformamide (DMF) and N,N-diisopropylethylamine (DIEA; 9.75 equiv) in Nmethylpyrrolidone (NMP). Double couplings were used for the syntheses with a coupling time of 2 h each. For Fmoc removal, piperidine-DMF (2:3) was used for 3 minutes, followed by 2×15 min deprotection with piperidine-DMF (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 RP-HPLC (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 ESI-MS (MSQ Plus Mass Spectrometer, Thermo). 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+. LL-37: 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 Scientific-Dionex Softron GmbH, Germering, Germany),


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Dionex Ultimate 3000 HPLC pump and Dionex Ultimate 3000 autosampler (Thermo ScientificDionex Softron GmbH, Germering, Germany), Shodex RI-101 refractive index detector (Shodex/Showa Denko Europe GmbH, Munich, Germany) and PSS’s ETA-2010 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(2-vinylpyridine) 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 25 min prior to measurement at a concentration of 1 mg/mL, filtered through a 0.45 µL 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. 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 96-well 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.

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 tris-buffered 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 96-well 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% TRITON-X 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): Hemolysis rate (%) =

1 − 13 x 100% (4) (133 − 13 )

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 LL-37 was evaluated by XTT assay using the human epithelial colorectal adenocarcinoma-derived cell line, Caco-2 (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. N-mono and N,N-dialkylation of 3,6-O-diTBDMS 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 mono-N-alkyl chitosan derivatives when 3,6-O-diTBDMS chitosan was used as the precursor.22 On the other hand, the conventional procedure for reductive mono- 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 mono-N-alkyl 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,N-dialkylated 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,N-dialkylated 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.

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 TBDMS-protected chitosan reaction, gave only around 65% di-substitution. This is comparable to the DS (0.5) of N,N-dibutyl chitosan16 obtained in earlier studies under acidic conditions. Thus, the use of TBDMS-protected chitosan is found to be necessary in order to obtain full N,Ndialkylation with the help of simple reaction conditions in organic solvent. The removal of the di-TBDMS 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) FT-IR spectra of (a) 3,6-O-diTBDMS chitosan, (b) N,Ndibutyl-3,6-O-diTBDMS chitosan and (c) N,N-dibutyl 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. Figure 1A shows a comparison of the 1H NMR spectra for the N,N-dibutyl 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 (H-9, H-9'), 1.32 (H-8, H8'), and 2.57 (H-7, H-7') 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 H-7 and H-7' protons in the butyl chains, which were initially merging with the H-2 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,N-dihexyl chitosan, (ii) N,N-diethyl chitosan, (iii) N,N-dibutyl chitosan, and (iv) N,N-dimethyl chitosan. with the H-2 peak) after deprotection. The strong correlation of H-7 protons with H-2 as well as the correlation between H-8, H-9, and H-10 protons in the COSY spectrum (not shown) also confirms the dibutylation of chitosan. Figure 1B shows a comparison of FT-IR spectra for the same reaction. Spectra (a) and (b) show stretching frequencies at 1361–1390 (m, C–H tert-butyl), 1255 (s, Si–CH3), and 778–837 (s, Si–CH3) cm-1, corresponding to the TBDMS group. The enhanced C–H stretching intensity in the 2871–2957 cm-1 region, accompanied by additional peaks at 1376 (C–H) and 1248 (C–N) cm1

in spectra (b) and (c), indicates the presence of the butyl group. The broad O–H band at 3430

cm-1 can be observed after the removal of the silyl groups in spectrum (c).


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An overlay of the 1H NMR spectra for the four dialkylated compounds, that is, N,N-dimethyl, N,N-diethyl, N,N-dibutyl, and N,N-dihexyl 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 mono-N-methylation;22 1.35 and 3.18–3.40 ppm correspond to the –CH3 and –CH2– protons, respectively, for the N,N-diethyl 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,N-dihexyl chitosan, which was insoluble in water; hence, the NMR spectrum was taken in CDCl3. The DS of each of the N,N-dialkyl derivative was calculated using equation (2), and the dialkylation was found to be 100% in each case. The presence of a sharp single H-1 peak in all spectra also indicates full dialkylation and the absence of any residual mono-alkylation. 3.1.2. Quaternization of the N-mono and N,N-dialkyl chitosan derivatives. The N-alkyl chitosan derivatives were quaternized by using methylating reagents. The mono-N-alkyl 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,N-trimethyl chitosan homopolymer was also synthesized from the TBDMS-protected chitosan using previous procedures.22 However, no product conversion was observed when these conditions were applied for the methylation of the N,N-dialkyl 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,N-dibutyl chitosan derivatives. Reaction/ starting material

Reagents

Temperature (°C)

Time (h)

DS (%)

Dialkylation/ Chitosan

Butyraldehyde/NaBH4/AcOH

40

96

65

Dialkylation/ 3,6-O-diTBDMS chitosan

Butyraldehyde/NaBH4/DCM

RT

96

100

Quaternization/ N,N-dibutyl-diTBDMS chitosan

DCM/Me2SO4

50

48

0

NMP/MeI/NaOH

50

48

0

DCM/CF3SO3Me

RT

48

30

NMP/MeI/Cs2CO3

50

48

0

DMF/H2O/MeI

RT

48

0

NMP/ MeI

40

48

20

NMP/MeI /NaOH*

40

48

75

Quaternization/ N,N-dibutyl chitosan

*This reaction was done twice. Methylation of the TBDMS-protected 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,N-dihexyl-3,6-OdiTBDMS chitosan could be observed (data not shown). In contrast, a high degree of methyl substitution was observed in the case of mono-N-alkyl chains. These results showed that the amino N atom in di-TBDMS 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/DMF-H2O33 and DMS/NaOH/NaCl/H2O34 did not result


22

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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,N-dibutyl-N-methyl chitosan,16 in which the degree of N,N-dialkylation 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,N-dialkyl derivatives with those of the mono-N-alkyl derivatives gave us a series of uniformly quaternized and highly water-soluble chitosan derivatives, which were suitable for antimicrobial testing. The 1H NMR spectrum of N,N-dibutyl-N-methyl chitosan shows a singlet appearing at 3.44 ppm clearly showing the N-methyl peak. The quaternization is also evident by the splitting of the H-1 proton and broadening of the dibutyl peak at 1.82 ppm in comparison to that of the N,Ndibutyl chitosan derivative (figure S17, supporting information) . Although quaternization of the unprotected chitosan leaves the possibility of some O-methylation during the reaction, the 1H NMR spectra did not show any prominent O-methyl peak (3.35 ppm)33 indicating N-methylation 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

Compound

Degree of Quaternisation (DQ%)

Isolated Yield (%)

Mw (Da)

MV(Da) [viscosity average Mw]

PDI

Solubility Aqueous

DMSO

N,N,N-trimethyl chitosan (3)

100

65

15730

15725

1.42

+++

–

N-ethyl-N,Ndimethylchitosan (5a)

66

45

9924

9919

1.03

+++

–

N-butyl-N,Ndimethylchitosan (5b)

74

43

8725

8721

1.02

+++

+/–

N-hexyl-N,Ndimethylchitosan (5c)

62

52

8766

8762

1.05

+++

++

N,N-diethyl-Nmethylchitosan (8b)

72

45

7875

7871

1.05

+++

–

N,N-dibutyl-Nmethylchitosan (8c)

75

42

9248

9245

1.01

+++

+/–

N,N-dihexyl-Nmethylchitosan (8d)

61

48

11774

1.23

+++

+++

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 cut-off 3000 Da) gives compounds having lower and uniform PDI values. 3.3. Antibacterial properties of the N-quaternary 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 LL-37, 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 surface-acting 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 LL-37, were synthesized and tested against the four different bacterial strains. The activity of melittin was found to be consistent with previous studies.41 LL-37 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,N-diethyl-N-methyl 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 (7-dilutions) than LL-37 (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,N-dihexyl-N-methyl chitosan (8d) and N-hexylN,N-dimethyl 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 LL-37, 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 chloride, cetyl pyridinium chloride, melittin, and LL-37. Gram positive bacteria

Gram negative bacteria

Hemolytic activity HC50 (µg/mL)

S.aureus MIC(µg/mL) (Selectivity HC50/MIC)

E.faecalis MIC(µg/mL) (Selectivity HC50 /MIC)

E.coli MIC(µg/mL) (Selectivity HC50 /MIC)

P.aeruginosa MIC(µg/mL) (Selectivity HC50 /MIC)

˃1024

32 (˃32)

128 (˃8)

64 (˃16)

512 (˃2)

˃1024

4 (˃256)

128 (˃8)

64 (˃16)

512 (˃2)

36

32 (1.12)

64 (0.56)

32 (1.12)

512 (0.07)

16

256 (0.06)

16 (1)

16 (1)

32 (0.5)

384

256 (1.5)

512 (0.75)

512 (0.75)

1024 (0.37)

20

512 (0.04)

128 (0.15)

32 (0.62)

512 (0.04)

40

1024 (0.04)

64 (0.62)

16 (2.5)

1024 (0.04)

Benzalkonium chloride

28

≤0.5 (≥56)

≤0.5 (≥56)

16 (1.75)

64 (0.43)

Cetyl pyridinium chloride

≤0.5

≤0.5 (~1)

≤0.5 (~1)

16 (256 for each compound, respectively. Compound 5a exhibited the highest selectivity amongst the chitosan derivatives, as well as the two peptides and the two disinfectants towards S.aureus. Compound 5c had the highest efficiency against E.coli and E.faecalis and exhibited a HC50 value of 16 µg/mL, giving it a low selectivity value of 1 against both the strains. Although 3 and 5a had higher MIC values (low activity) against E.faecalis, owing to their non-hemolytic properties, they displayed much higher selectivity (>8) than 5c. Highly active derivatives 3 and 5a against E.coli displayed very good selectivity of ˃16, as compared to derivatives 5c and 8d (with selectivities of 1 and 2.5 respectively). The two derivatives 3 and 5a were also found to have considerably higher selectivity than CPC, melittin, and LL-37 against E.faecalis and E.coli. Against P.aeruginosa, all the derivatives were seen to possess similar antibacterial activity, while their selectivity values


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were found to be in the range 0.04–2. Thus, amongst all of the chitosan derivatives, 3 and 5a were found to have excellent selectivity towards all four bacterial strains over mammalian cells. The cytotoxicity of the compounds towards the Caco-2 human epithelial cell lines (Figure 3) also showed that the shorter alkyl chain derivatives were lower in toxicity, and the toxicity increased with increase in the length of the alkyl chains in both the N-monoalkyl and the N,Ndialkyl series of quaternary chitosan derivatives, which is consistent with the findings from the RBC assay. This result can be further verified by comparison with our previous findings where cytotoxicity towards human bronchial epithelial cell VA10 for the quaternary N-alkyl chitosan derivatives was found to increase with increase in the alkyl chain length.27 A comparison of the toxicity data for the chitosan derivatives with that of melittin and LL-37 towards VA10 cells showed that TMC (3) and N-ethyl-N,N-dimethyl chitosan (5a), showed relatively lower toxicity than melittin and comparable toxicity as LL-37.

4. CONCLUSION The antimicrobial properties of chitosan and of chitosan derivatives represent an active research field, with more than 100 original publications in the last year.54-59 A large number of different chitosan derivatives with notable antimicrobial properties have been reported.60-64 However, there are few systematic studies for determining structure–activity relationships and for the contribution of chemical modification towards activity. The current work focused on quaternary N-alkyl derivatives of chitosan, which have been intensively studied as promising antimicrobial agents, but these studies have not agreed in terms of the structural features that contribute to optimal activity. One of the reasons for this could be the partial N-alkylation in many of the reported derivatives.


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In the first part of the study, TBDMS chitosan was utilized to achieve complete conversion to obtain 100%-dialkylated derivatives of chitosan with long alkyl chains and we have also obtained a high degree of quaternization (through N-methylation) of these derivatives. In the second part of the study, these well-defined derivatives could be used to develop a clear relationship between the length of the alkyl chain and antibacterial activity, but optimal structure depended on the type of bacterium. Short alkyl chain derivatives showed high activity against S.aureus, whereas more hydrophobic N-hexyl derivatives were active against E.coli and E.faecalis. Their activity against P.aeruginosa was independent of any variation in chain length and the activity was mainly dependent on the cationic charge that was present. All the derivatives displayed bactericidal properties even against the highly resistant Gram positive E.faecalis and Gram negative P.aeruginosa. Highly selective compounds, which were significantly more active against bacteria than human cells could be obtained. The quaternary polymers with mono-methyl and mono-ethyl chains thus exhibited significantly higher selectivity than the disinfectant CPC as well as the two antimicrobial peptides melittin and LL-37 in most cases and hence can be regarded as promising development as antibacterial agents.

ASSOCIATED CONTENT Supporting Information. 1H NMR spectra of chitosan intermediate and final derivatives; COSY-NMR spectra of final chitosan compounds 3, 8b, 8c and 8d; FT-IR spectra of chitosan intermediate and final compounds; Solubility studies for the chitosan derivatives at different time intervals, MLC values for final chitosan derivatives, CPC, BAC, melittin and LL-37; graph showing hemolysis rate for the final chitosan derivatives, EC50 values for the N,N-dialkyl-Nmethyl chitosan derivatives towards Caco-2 cell line, results of previous investigation of


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cytotoxicity of N-alkyl-N,N-dimethyl chitosan derivatives towards bronchial epithelial cell line, VA10, Cytotoxicity of the peptides LL-37 and Melittin towards bronchial epithelial cell line,VA10, Cytotoxicity (experimental). This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author * Tel.: +354-8228301; Fax: +354-5254071. E-mail: [email protected]. Author contributions M.M. and M.H. conceived the study, which was planned in collaboration with P.S. P.S. synthesized and characterized compounds 3, 5a–c, and 8b–d (Scheme-1). B.E.B. synthesized melittin and LL-37 in collaboration with K.K.S., M.B.T. and K.J.J. P.S. and M.H. preformed the antibacterial study. P.S. and O.E.S. measured the hemolytic activity and O.E.S did the investigation of cytotoxicity towards Caco-2 cells. The first draft was written by P.S. and M.M. All authors participated in interpreting the results and preparing the final version of the manuscript.

ACKNOWLEDGMENT This research was supported by the Icelandic Research Fund (Grant 120443021) and doctoral grants from the University of Iceland Research Fund. Additional support provided by Bergþóru og Þorsteins Schevings Thorsteinsson is highly acknowledged. The chitosan polymer used in this study was kindly donated by Genis ehf. We like to thank Professor Thorarinn Gudjonsson,


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Faculty of Medicine, University of Iceland to allow us to include results from our previous study, done in his lab, on the cytotoxicity towards VA10 cells.

ABBREVIATIONS DS, degree of substitution; TBDMS, tert-butyldimethylsilyl; Mw, average molecular weight; DA, degree of acetylation; NMP, N-methyl-2-pyrrolidone; TBAF, tetrabutylammoniumflouride; DMF, N,N-dimethylformamide; DIEA, N,N-diisopropylethylamine; TFA, triflouroacetic acid; TES, triethylsilane; HOAt, 1-hydroxy-7-azabenzotriazole; HBTU, 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate; GPC, gel permeation chromatography; PSS, Polymer Standards Service; PDI, polydispersity index; ATCC, American type culture collection; S.aureus, Staphylococcus aureus; E.faecalis, Enterococcus faecalis; E.coli, Escherichia coli; P.aeruginosa, Pseudomonas aeruginosa; MIC, minimum inhibitory concentration; MLC, minimum lethal concentration; RBC, red blood cell; TBS, tris-buffered saline; DQ, degree of quaternizaton; MV, viscosity average Mw; DMSO, dimethyl sulfoxide; CPC, cetylpyridinium chloride; BAC, benzalkonium chloride; AMP, antimicrobial peptide.

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For Table of Contents Use Only

The Impact of Chain Length on Antibacterial Activity and Hemocompatibility of Quaternary N-Alkyl and N,N-Dialkyl Chitosan Derivatives

Priyanka Sahariah, Berglind E. Benediktssdóttir, Martha Á. Hjálmarsdóttir, Olafur E. Sigurjonsson, Kasper K. Sørensen, Mikkel B. Thygesen, Knud J. Jensen, Már Másson*


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Impact of Chain Length on Antibacterial Activity ... - ACS Publications

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