The Effect of Substituent, Degree of Acetylation and Positioning of the Cationic Charge on the Antibacterial Activity of Quaternary Chitosan Derivatives

A series of water-soluble cationic chitosan derivatives were prepared by chemoselective functionalization at the amino group of five different parent chitosans having varying degrees of acetylation and molecular weight. The quaternary moieties were introduced at different alkyl spacer lengths from the polymer backbone (C-0, C-2 and C-6) with the aid of 3,6-di-O-tert-butyldimethylsilyl protection of the chitosan backbone, thus allowing full (100%) substitution of the free amino groups. All of the derivatives were characterized using 1H-NMR, 1H-1H COSY and FT-IR spectroscopy, while molecular weight was determined by GPC. Antibacterial activity was investigated against Gram positive S. aureus and Gram negative E. coli. The relationship between structure and activity/toxicity was defined, considering the effect of the cationic group’s structure and its distance from the polymer backbone, as well as the degree of acetylation within a molecular weight range of 7–23 kDa for the final compounds. The N,N,N-trimethyl chitosan with 100% quaternization showed the highest antibacterial activity with moderate cytotoxicity, while increasing the spacer length reduced the activity. Trimethylammoniumyl quaternary ammonium moieties contributed more to activity than 1-pyridiniumyl moieties. In general, no trend in the antibacterial activity of the compounds with increasing molecular weight or degree of acetylation up to 34% was observed.


Chitosan Mesylate (Mes-CS) (2 i-v )
Free base chitosan (1 i-v ) (1 g, 6 mmol) was suspended in methanesulfonic acid (CH 3 SO 3 H) (10 mL, 0.153 mol) and cooled to 10 °C. To the reaction mixture, H 2 O (~10 mL) was added dropwise until a clear homogeneous solution was obtained. The reaction mixture was then stirred for 1 h before precipitating with EtOH (40 mL) resulting in a gel-like precipitate. The precipitate was filtered under suction using a sintered funnel and washed with EtOH (3 × 25 mL), followed by washing with acetone (3 × 20 mL). The material was then allowed to air-dry for 1 h. This salt precipitate was redissolved in a minimum quantity of H 2 O (5-10 mL) and reprecipitated using acetone (60 mL), filtered, washed with acetone (2 × 30 mL) and the obtained material further dried in a vacuum oven at 40 °C overnight to afford corresponding finely powdered off-white chitosan mesylate salt (2 i-v ) (1.39 g, 90%). FT

3,6-di-O-tert-Butyldimethylsilyl-chitosan (diTBDMS-CS) (3 i-v )
Chitosan mesylate (2 i-v ) (1 g, 3.97 mmol) was dissolved in dry DMSO (15 mL) under N 2 atmosphere. To this reaction mixture, imidazole (2.71 g, 39.74 mmol) and TBDMSCl (2.99 g, 19.87 mmol) in dry DMSO (13 mL) wereadded dropwise, and the resulting mixture was stirred at 25 °C. During the addition of the reagents, the reaction mixture turned cloudy, and eventually, sometime after completion of the addition, a solid gel-type material separated out from the solution. The reaction mixture was stirred for 24 h at 25 °C and then filtered by using a sintered funnel, and the solid obtained was continuously triturated while washing with H 2 O (5 × 30 mL), followed by washing with CH 3 CN (3 × 20 mL). The material was air dried and then further dried in a vacuum oven at 40 °C overnight, to afford corresponding off-white finely powdered silyl protected DiTBDMS-CS compound (3 i-v ) (1.46 g, 96%).

N-(Bromoacetyl)-3,6-di-O-TBDMS-chitosan (BrA-diTBDMS-CS) (4 i-v )
Silyl chitosan 3 i-v (1 g, 2.6 mmol) was dissolved in dry CH 2 Cl 2 (15 mL) under N 2 atmosphere. The reaction mixture was cooled to −20 °C by using a salt-ice cooling mixture. To the reaction mixture, Et 3 N (1.81 mL, 13 mmol) was added, followed by the slow dropwise addition of bromoacetyl bromide (0.91 mL, 10 mmol). Stirring was continued for 1 h at a constant temperature maintained at −20 °C before the reaction mixture was diluted with CH 2 Cl 2 (30 mL) and concentrated in vacuo. The crude material obtained was triturated and stirred in CH 3 CN (15 mL), filtered and washed with CH 3 CN (3 × 15 mL) and air-dried. Dry material was redissolved in CH 2 Cl 2 (45 mL) and washed with H 2 O (3 × 30 mL) and brine (20 mL). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated in vacuo to afford corresponding pale yellow powdered bromoacetyl intermediate

N-(2-(N,N,N-Trimethylammoniumyl)acetyl)-3,6-di-O-TBDMS-chitosan Bromide (5 i-v )
Freshly prepared bromoacyl intermediate 4 i-v (450 mg, 0.9 mmol) was dissolved in CH 2 Cl 2 (10 mL) under N 2 atmosphere. Excess Me 3 N (4.2 molar in EtOH) (10 mL) was added, and the resulting mixture was stirred for 24 h at 25 °C. The reaction mixture was concentrated in vacuo to isolate the corresponding crude Product 5 i-v . These crude materials were used directly for the next deprotection step without any purification and characterization.

hexanoyl)-3,6-di-O-TBDMS-chitosan Bromide/Iodide (10 i-v )
Freshly prepared bromohexanoyl compound (9 i-v ) (450 mg, 0.8 mmol) was dissolved in CH 2 Cl 2 (12 mL) under N 2 atmosphere. To the reaction mixture, excess Me 3 N (4.2 molar in EtOH) (15 mL) was added, and the resulting mixture was stirred for 24 h at 25 °C. Some white precipitate was formed as the reaction progressed, and this increased after the addition of a catalytic amount of KI. The stirring was continued further for 24 h. The reaction mixture was concentrated in vacuo to isolate the corresponding crude Product 10 i-v . These crude materials were washed with diethyl ether, filtered, dried and then used directly for the next deprotection stage without any further purification or characterization.

N-(6-(1-Pyridiniumyl)hexanoyl)-3,6-di-O-TBDMS-chitosan Bromide/Iodide (12 i-v )
Freshly prepared bromohexanoyl compound (9 i-v ) (400 mg, 0.71 mmol) was dissolved in CH 2 Cl 2 (5 mL) under N 2 atmosphere. To the reaction mixture, excess quantity of pyridine (15 mL) and catalytic amount of KI was added, and the resulting mixture continued to be stirred for 48 h at 25 °C. The reaction mixture was concentrated completely in vacuo to isolate the corresponding crude product (12 i-v ). These crude materials were washed with diethyl ether, filtered, dried and then used directly for the next deprotection stage without any purification and characterization.

General TBDMS Deprotection Procedure to Give the Final Quaternary
AmmoniumylandPyridiniumylDerivatives (6 i-v , 8 i-v , 11 i-v , 13 The compounds (5 i-v , 7 i-v , 10 i-v or 12 i-v ) (300 mg)weredissolved in MeOH (4-5 mL), and concHCl (1-2 mL) was added. The reaction mixture was stirred for 12 h at 25 °C, diluted with H 2 O (10 mL) and, then, ion exchanged by adding 10% NaCl (aqueous) (w/v) (15 mL), and the resulting mixture was stirred for 1 h at 25 °C. The colorless solution was then dialyzed against 5% aqueous NaCl solution for oneday and then against deionized water for twodays, before it was freeze-dried to afford the corresponding deprotectedwater-soluble, white, fluffy quaternized product (6 i-v , 8 i-v , 11 i-v or 13 i-v ). The deprotection was repeated if needed to remove traces of silyl impurities that could be observed in 1 H NMR after the first round.