Novel amphiphilic dextran esters with antimicrobial activity

Journal Pre-proof Novel amphiphilic dextran esters with antimicrobial activity

Magdalena Cristina Stanciu, Dalila Belei, Elena Bicu, Cristina G. Tuchilus, Marieta Nichifor PII:

S0141-8130(19)39351-1

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.021

Reference:

BIOMAC 14637

To appear in:

International Journal of Biological Macromolecules

Received date:

15 November 2019

Revised date:

9 January 2020

Accepted date:

3 February 2020

Please cite this article as: M.C. Stanciu, D. Belei, E. Bicu, et al., Novel amphiphilic dextran esters with antimicrobial activity, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.02.021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof Novel amphiphilic dextran esters with antimicrobial activity Magdalena Cristina Stanciua*, Dalila Beleib, Elena Bicub, Cristina G. Tuchilusc and Marieta Nichifora a

“Petru Poni” Institute of Macromolecular Chemistry, Department of Natural Polymers, Bioactive and Biocompatible Materials, Gr. Ghica Voda Alley, no 41 A, 700457, Iasi, Romania

b

“Al. I. Cuza” University of Iasi, Faculty of Chemistry, Department of Organic Chemistry, Bd. Carol I, no 11, 700506, Iasi, Romania “Grigore T. Popa” University of Medicine and Pharmacy, Department of Microbiology,

of

c

ur

na

lP

re

-p

ro

Faculty of Medicine, University Street, no 16, 700115, Iaşi, Romania

Jo

* Corresponding author

Magdalena Cristina Stanciu

“Petru Poni” Institute of Macromolecular Chemistry Department of Natural Polymers, Bioactive and Biocompatible Materials Grigore Ghica Voda Alley, no 41 A, 700487, Iasi, Romania e-mail: [email protected] FAX: +40232 21129

1

Journal Pre-proof Abstract New amphiphilic dextran esters were obtained by polysaccharide functionalization with different substituted 1,2,3-triazoles-4-carboxylic acid via in situ activation with N, N’carbonyldiimidazole. Nitrogen-containing heterocyclic derivatives were achieved by copper(I)-catalyzed cycloaddition reaction between organic azides and ethyl propiolate. Structural characteristics of the compounds were studied by elemental analysis, Fourier transform infrared and nuclear magnetic resonance spectroscopy (1H and

13

C-NMR).

Thermogravimetric analysis, differential scanning calorimetry and wide-angle X-ray diffraction were used for esters characterization. Properties of polymeric self-associates,

of

formed in aqueous solution, were studied by dynamic light scattering and transmission

ro

electron microscopy. The critical aggregation concentration values for dextran esters, determined by fluorescence spectroscopy, were in the range of 4.1-9.5 mg/dL. Antimicrobial

-p

activity, investigated for some of the polymers by disc-diffusion method, pointed out that

lP

re

polysaccharide esters were active.

Jo

ur

antimicrobial activity

na

Keywords: dextran; 1,4-disubstituted-1,2,3-triazole; amphiphilic esters; self-association;

2

Journal Pre-proof 1. Introduction Natural polysaccharides and their derivatives have been intensively studied over the last decades because they are inexpensive, non-toxic, readily available and biodegradable. Chemical modification is a way to introduce desired properties in biopolymers for specific applications with a minimum loss in their native characteristics. Dextran, a neutral extracellular bacterial polysaccharide composed of D-glucopyranose units linked by linear α1,6 glycosidic bonds with a low degree of α-1,3-linked side chains, has been widely explored as a biomaterial since it is biocompatible and non-toxic which justify its use as drug carrier, plasma volume expander, antithrombotic and anti-inflammator [1]. Many dextran esters were

of

obtained by reaction of the biopolymer with activated carboxylic acids. N, N’-

ro

carbonyldiimidazole (CDI), a dialkylcarbodiimide used as activating reagent, is suitable for the derivatization of the polysaccharide because it affords homogeneous one-pot synthesis.

-p

The use of CDI avoids most of the side reactions because during conversion the reactive imidazolide of the acid and two non-toxic by-products (CO2 and imidazole) are generated [2].

re

Thus, many dextran esters with randomly distributed side chains derived from: pharmaceutical agents (cromoglycic acid [3], naproxen [4,5], daunorubicin [6], mitomycin C

lP

[7], cisplatin [8], ibuprofen [5], ketoprofen [9], 5-aminosalicylic acid [10], celecoxib [11], nalidixic acid [12], etc) or azobenzene, cinnamic and acetic acid derivatives [13] were

na

prepared using CDI as activating agent. N, N’-carbonyldiimidazole was also used for the esterification of other polysaccharides, such as: starch, cellulose, pullulan. Thus, starch was

ur

esterified with fatty acids [14] and piperic acid [15]. Cellulose esters were obtained by using either carboxylic acids with chiral, heterocyclic, crown ether and cyclodextrin containing

Jo

moieties [16] or adamantane carboxylic acid [17]. Pullulan was esterified with aspirin [18] and abietic acid [19]. Amphiphilic polysaccharide esters have the ability to self-assemble in aqueous solution. Size and morphology of the self-aggregates is mainly influenced by the chemical structure of the functionalized polysaccharides and their degree of substitution (DS)[5,13,18]. Applications predicted for these self-assembling polymers include: drug delivery systems [5-12,15,18], antimicrobial agents [17], light sensitive materials [13], smart materials for chiral and selective separation processes [16]. 1,2,3-Triazole and its derivatives are an important class of five-membered aromatic ring Nheterocycles, generally prepared via CuAAC reaction between an azide and a terminal alkyne [20]. The heterocyclic 1,2,3-triazole ring is one of the key structural elements in many drug molecules and polymers. Although 1,2,3‐ triazole does not occur in nature, the synthetic molecules containing 1,2,3-triazole moieties have an important role in medicinal chemistry 3

Journal Pre-proof due to their therapeutic properties and good metabolic stability. Thus, many 1,2,3-triazoles proved various biological activities, such as: anticancer [21, 22], antimicrobial [23, 24], antimalarial [25], anticonvulsant [26], antiviral [27] analgesic [28], anti-HIV [29], antitubercular [30]. In addition to this, 1,2,3-triazoles have found broad applications as corrosion inhibitors [31] and photostabilizers [32, 33] or in agriculture as fungicides [34] and plant growth regulators [35]. In the present paper, we report the synthesis of novel amphiphilic esters based on dextran which were obtained by a one-pot procedure based on the reaction between the polysaccharide and different substituted 1,2,3-triazoles-4-carboxylates activated in situ with

of

CDI. 1,2,3-Triazole derivatives were achieved by copper(I)-catalyzed Huisgen reaction.

ro

Chemical modification of dextran was carried out in order to obtain hydrophobically modified polymers able to self-assemble in aqueous solution. Capacity to form micelles and

-p

their characteristics were examined using fluorescence technique, dynamic light scattering and transmission electron microscopy. Thermal stability of polysaccharide-based polymers

re

was also studied and their amorphous structure was pointed out. The antibacterial and antifungal activity for some of these new polymers was tested using several bacterial and

2.1. Materials

na

2. Materials and methods

lP

yeast strains.

ur

Dextran from Leuconostoc spp (Mr = 40,000 Da, as reported by supplier), N, N’carbonyldiimidazole, ethyl propiolate, sodium L-ascorbate, copper(II) sulfate pentahydrate

Jo

were purchased from Sigma-Aldrich and used as received. The other reagents of analytical grade were from Sigma and were purified/dried by standard methods. Dialysis membrane (MWCO 12,000) was bought from Sigma-Aldrich.

2.2. Synthetic procedures 2,2,1, General procedure for the synthesis of ethyl 1H-1,2,3-triazole-4-carboxylates (2a–f) Ethyl 1H-1,2,3-triazole-4-carboxylates (Scheme 1) were obtained by modifying the protocol of click reaction between azide derivatives 1a-f and ethyl propiolate, the yields being higher than those previously reported [36]. Scheme 1 The synthesis of compound 2a is presented as an example.

4

Journal Pre-proof 0.12 mL (1.2 mmole) ethyl propiolate was added to 0.16 g (1 mmole) 2-azido-1-phenylethanone (1a) dissolved in a solvent mixture of 6 mL t-BuOH and 1.5 mL MeOH . When the mixture was cleared, 1 mL of sodium L-ascorbate 10 % aqueous solution and 1 mL of copper sulfate 3% aqueous solution were added. The reaction mixture was kept for 24 h under magnetic stirring at 50 °C. Cold water and ammonium hydroxyde were added into the reaction flask after its cooling to room temperature. The resulting suspension was vigorously stirred to complete the triazole precipitation. The obtained solid was separated by filtration, washed with ethyl acetate and dried under vacuum. Other ethyl 1H-1,2,3-triazole-4carboxylates (2b-f) were prepared by the same procedure as for 2a.

of

The carbon atoms of 2a–f are numbered according to Scheme 1. For 2e and 2f there is an

ro

additional carbon atom, namely C13, coming from methyl (2e) and methoxy group (2f),

-p

respectively, which are bound in para position of the benzene ring.

Ethyl 1-(2-phenyl-2-oxoethyl)-1H-1,2,3-triazole-4-carboxylate (2a)

re

white solid, yield: 0.21 g (81 %); mp (EtOAc) 174-175 °C [37]. FTIR (KBr)(cm-1): 1725 (νC=O ester), 1703 (νC=O ketone), 1601 (νC=C triazole), 1450 (νN=N arom), 1046 (νC-N); 1H-NMR (DMSO-

lP

d6, 400 MHz) δ (ppm): 1.31 (t, J = 6.9 Hz, 3H, CbH3), 4.34 (q, J =6.9 Hz, 2H, CaH2), 6.28 (s, 2H, C7H2), 7.62 (m, 1H, C11H), 7.75 (m, 1H, C12H), 8.08 (d, J = 7.9 Hz, 1H, C10H), 8.72 (s, 13

C NMR (DMSO-d6, 100 MHz):  (ppm): 14.9 (Cb), 56.9 (C7), 61.3 (Ca), 128.9

na

1H, C5H);

ur

(C11), 129.8 (C10), 131.5 (C5), 135.6 (C12), 138.7 (C4), 139.6 (C9), 161.3 (C6), 192.4 (C8).

Ethyl 1-[2-(4-fluoro-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (2b)

Jo

white solid, yield: 0.22 g (80 %); mp (EtOAc) 178-180 °C [37]. FTIR (KBr) (cm-1): 1726 (νC=O ester), 1707 (νC=O ketone), 1599 (νC=C triazole), 1450 (νN=N arom), 1229 (νC-F), 1057 (νC-N);

1

H-

NMR (DMSO-d6, 400 MHz) δ (ppm): 1.33 (t, J = 6.8 Hz, 3H, CbH3), 4.33 (q, J =6.8 Hz, 2H, CaH2), 6.27 (s, 2H, C7H2), 7.45 (d, J=8.8 Hz, 1H, C11H), 8.18 (d, J = 6.4 Hz, 1H, C10H), 8.71 (s, 1H, C5H); 13C NMR (DMSO-d6, 100 MHz)  (ppm): 14.0 (Cb), 56.1 (C7), 60.6 (Ca), 116.1 (C11), 130.6 (C5), 131.3 (C10), 139.7 (C4), 144 (C9), 160.3 (C6), 164.3 (C12), 190.2 (C8).

Ethyl 1-[2-(4-chloro-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (2c) beige solid, yield: 0.31 g (46 %); mp (EtOAc) 192-193 °C [37]. FTIR (KBr)(cm-1): 1720 (νC=O ester), 1694 (νC=O ketone), 1564 (νC=C triazole), 1475 (νN=N arom), 1089 (νC-Cl), 1025 (νC-N); 1HNMR (DMSO-d6, 400 MHz) δ (ppm): 1.31 (t, J = 7.2 Hz, 3H, CbH3), 4.34 (q, J =7.2 Hz, 2H, 5

Journal Pre-proof CaH2), 6.26 (s, 2H, C7H2), 7.71 (d, J = 8.4 Hz, 1H, C11H), 8.10 (d, J = 8.8 Hz, 1H, C10H), 8.70 (s, 1H, C5H); 13C NMR (DMSO-d6, 100 MHz)  (ppm): 14.1 (Cb), 56.0 (C7), 60.4 (Ca), 129.1 (C11), 130.2 (C10), 130.6 (C5), 132.6 (C12), 139 (C4), 139.5 (C9), 160.3 (C6), 190.7 (C8).

Ethyl 1-[2-(4-bromo-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (2d) beige solid, yield: 0.28 g (83 %); mp (EtOAc) 196-198 °C [37]. FTIR (KBr)(cm-1): 1724 (νC=O ester), 1684 (νC=O ketone), 1586 (νC=C triazole), 1437 (νN=N arom), 1053 (νC-Br), 1025 (νC-N); 1HNMR (DMSO-d6, 400 MHz) δ (ppm): 1.32 (t, J = 7.2 Hz, 3H, CbH3), 4.31 (q, J =7.2 Hz, 2H, CaH2), 6.24 (s, 2H, C7H2), 7.70 (d, J = 8.4 Hz, 1H, C11H), 8.11 (d, J = 8.8 Hz, 1H, C10H), 8.71

of

(s, 1H, C5H); 13C NMR (DMSO-d6, 100 MHz)  (ppm): 14.3 (Cb), 56.1 (C7), 60.4 (Ca), 128.7

ro

(C12), 130.4 (C10), 130.5 (C5), 132.6 (C11), 133 (C4), 139.7 (C9), 160.5 (C6), 190.6 (C8).

-p

Ethyl 1-[2-(4-methyl-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (2e) yellow solid, yield: 0.21 g (77 %); mp (EtOAc) 187-188 °C [37]. FTIR (KBr) (cm-1): 1721

re

(νC=O ester), 1694 (νC=O ketone), 1611 (νC=C triazole), 1435 (νN=N arom), 1019 (νC-N); 1H-NMR (DMSOd6, 400 MHz) δ (ppm): 1.33 (t, J = 6.8 Hz, 3H, CbH3), 2.43 (s, 3H, C13H3), 4.32 (q, J =6.8 Hz,

lP

2H, CaH2), 6.24 (s, 2H, C7H2), 7.43 (d, J = 8.0 Hz, 1H, C11H), 7.98 (d, J = 7.9 Hz, 1H, C10H), 8.72 (s, 1H, C5H); 13C NMR (DMSO-d6, 100 MHz)  (ppm): 14.1 (Cb), 21.3 (C13), 55.9 (C7),

na

60.4 (Ca), 128.2 (C10), 129.5 (C11), 130.8 (C5), 131.3 (C12), 139.6 (C4), 145.0 (C9), 160.2 (C6),

ur

191.0 (C8).

Jo

Ethyl 1-[2-(4-methoxy-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylate (2f) white solid, yield: 0.24 g (84 %); mp (EtOAc) 170-172 °C [37]. FTIR (KBr)(cm-1): 1722 (νC=O ester), 1680 (νC=O ketone), 1605 (νC=C triazole), 1434 (νN=N arom), 1033 (νC-N); 1H-NMR (DMSOd6, 400 MHz) δ (ppm): 1.33 (t, J = 6.8 Hz, 3H, CbH3), 3.75 (s, 3H, C13H3), 4.32 (q, J =6.8 Hz, 2H, CaH2), 6.24 (s, 2H, C7H2), 7.45 (d, J = 8.0 Hz, 1H, C11H), 7.97 (d, J = 8.0 Hz, 1H, C10H), 8.71 (s, 1H, C5H);

C NMR (DMSO-d6 , 100 MHz)  (ppm): 14.1 (Cb), 55.9 (C7), 56.4

13

(C13H3), 60.4 (Ca), 128.3 (C11), 129.4 (C10), 131.0 (C5), 139.2 (C4), 139.8 (C9), 158.8 (C12), 160.2 (C6), 191.0 (C8).

2.2.2. General procedure for the synthesis of 1H-1,2,3-triazole-4-carboxylates (3a–f) 1-[2-(4-A-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acids (3a-f) were synthesized by hydrolysis of esters 2a-f (Scheme 1). The experimental procedure used for hydrolysis 6

Journal Pre-proof reaction was modified compared to the one previously reported for this synthesis [36] to get better yields. The reaction protocol for obtaining 3a is presented as an example. 1 mL aqueous solution (30 %) KOH was added to a suspension of 0.26 g (1 mmole) ethyl 1H-1,2,3-triazole-4-carboxylate (2a) in 13 mL methanol. The reaction mixture was heated under reflux for 1 hour. After cooling to room temperature, the pH was adjusted to 4 by adding 0.1M HCl aqueous solution. The resulting solid was filtered, washed with ethanol and then dried under vacuum. Other 1H-1,2,3-triazole-4-carboxylates (3b-f) were prepared by the same procedure as for 3a.

of

1-(2-phenyl-2-oxoethyl)-1H-1,2,3-triazole-4-carboxylic acid (3a)

white solid, yield: 0.2 g (88 %); mp (EtOH) 220-221 °C; FTIR (KBr) (cm-1): 3120 (νO-H acid), ketone

), 1681 (νC=O

acid

), 1612 (νC=C

triazole

), 1429 (νN=N

ro

1703 (νC=O

arom),

1043 (νC-N); 1H-NMR

-p

(DMSO-d6, 400 MHz) δ (ppm): 6.20 (s, 2H, C7H2 ), 7.37 (d, J = 7.8 Hz, 1H, C11H), 7.45 (t, J = 7.2 Hz, 1H, C12H), 7.86 (d, J = 7.8 Hz, 1H, C10H), 8.58 (s, 1H, C5H), 13.16 (s, 1H,

re

C6OOH). 13C NMR (DMSO-d6, 100 MHz):  (ppm) 56.3 (C7), 128.4 (C11), 130.1 (C10), 131.4

lP

(C5), 135.8 (C12), 139.1 (C4), 141.2 (C9), 161.1 (C6), 192 (C8).

1-[2-(4-Fluoro-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (3b)

na

white solid, yield 0.24 g (98 %); mp (EtOH) 223–224 °C. FTIR (KBr)(cm-1): 3121 (νO-H acid), 1709 (νC=O ketone),1698 (νC=O acid), 1612 (νC=C triazole), 1429 (νN=N arom), 1227 (νC-F), 1055 (νC-N); 1H-

ur

NMR (DMSO-d6, 400 MHz) δ (ppm): 6.25 (s, 2H, C7H2 ), 7.46 (d, J = 8.6 Hz, 1H, C11H), 8.62 (d, J = 8.6 Hz, 1H, C10H), 8.64 (s, 1H, C5H), 13.0 (s, 1H, C6OOH). 13C NMR (DMSO-

Jo

d6, 100 MHz)  (ppm): 56.5 (C7), 116.6 (C11), 131.1 (C5), 131.8 (C10), 140.2 (C4), 144 (C9), 160.8 (C6), 164.7 (C12), 190.8 (C8).

1-[2-(4-Chloro-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (3c) white solid, yield: 0.23 g (88 %); mp (EtOH) 236–238 °C. FTIR (KBr)(cm-1): 3121 (νO-H acid),

1709 (νC=O ketone), 1682 (νC=O acid), 1589 (νC=C triazole), 1438 (νN=N arom), 1092 (νC-Cl), 990 (νC-

1

H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.26 (s, 2H, C7H2), 7.72 (d, J = 8.6 Hz, 1H,

N);

C11H), 8.09 (d, J = 8.6 Hz, 1H, C10H), 8.60 (s, 1H, C5H), 13.25 (s, 1H, C6OOH). 13C NMR (DMSO-d6, 100 MHz)  (ppm): 56.6 (C7), 129.5 (C11), 130.6 (C10), 131.0 (C5), 133.0 (C12), 139.7 (C4), 140.4 (C9), 160.3 (C6), 191.2 (C8).

7

Journal Pre-proof 1-[2-(4-Bromo-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (3d) white solid, yield: 0.29 g (92 %); mp (EtOH) 241–243 °C. FTIR (KBr)(cm-1): 3121 (νO-H acid),

1691 (νC=O ketone), 1682 (νC=O acid), 1586 (νC=C triazole), 1431 (νN=N arom), 1071 (νC-Br), 1025 (νC-

1

H-NMR (DMSO-d6, 400 MHz) δ (ppm): 6.24 (s, 2H, C7H2), 7.85 (d, J = 8.8 Hz, 1H,

N);

C11H), 8.00 (d, J = 8.8 Hz, 1H, C10H), 8.61 (s, 1H, C5H), 13.14 (s, 1H, C6OOH).

13

C NMR

(DMSO-d6, 100 MHz)  (ppm) : 56.5 (C7), 129.0 (C12), 130.6 (C10), 131.0 (C5), 132.6 (C11), 133.4 (C4), 140.4 (C9), 160.1 (C6), 190.8 (C8).

1-[2-(4-Methyl-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (3e)

acid),

of

yellow solid, yield: 0.22 g (89 %); mp (EtOH) 235–237 °C. FTIR (KBr)(cm-1): 3120 (νO-H 1696 (νC=O ketone), 1676 (νC=O acid), 1604 (νC=C triazole), 1439 (νN=N arom), 1055 (νC-N); 1H-NMR

ro

(DMSO-d6, 400 MHz) δ (ppm): 2.42 (s, 3H, C13H3 ), 6.22 (s, 2H, C7H2), 7.43 (d, J = 8.2 Hz, 13

C

-p

1H, C11H), 7.98 (d, J = 8.2 Hz, 1H, C10H), 8.62 (s, 1H, C5H), 13.16 (s, 1H, C6OOH).

NMR (DMSO-d6, 100 MHz)  (ppm): 21.7 (C13), 56.4 (C7), 128.8 (C10), 130.0 (C11), 131.1

lP

re

(C5), 131.9 (C12), 140.1 (C4), 145.4 (C9), 160.2 (C6), 191.6 (C8).

1-[2-(4-Methoxy-phenyl)-2-oxoethyl]-1H-1,2,3-triazole-4-carboxylic acid (3f) yellow solid, yield: 0.29 g (87 %); mp (EtOH) 218–220 °C. FTIR (KBr)(cm-1): 3118 (νO-H 1707 (νC=O ketone), 1681 (νC=O acid), 1604 (νC=C triazole), 1425 (νN=N arom), 1038 (νC-N); 1H-NMR

na

acid),

(DMSO-d6, 400 MHz) δ (ppm): 3.73 (s, 3H, C13H3), 6.22 (s, 2H, C7H2), 7.46 (d, J = 8.2 Hz,

ur

1H, C11H), 7.97 (d, J = 8.2 Hz, 1H, C10H), 8.64 (s, 1H, C5H), 13.20 (s, 1H, C6OOH).

13

C

Jo

NMR (DMSO-d6, 100 MHz)  (ppm): 56.4 (C7), 58.7 (C13), 128.8 (C11), 130.0 (C10), 131.1 (C5), 131.9 (C4), 140.1 (C9), 158,4 (C12), 160.2 (C6), 191.3 (C8).

2.2.3. General procedure for the synthesis of dextran esters (Dex40-T-A)(4a-f) As a general procedure, the synthesis of dextran derivatives (Scheme 1) was carried out by esterification of the polysaccharide with compounds 3a-f activated in situ with CDI. The preparation of dextran ester 4a is presented as an example. 0.6 g (3.72 mmol) CDI was added to 0.86 g (3.72 mmol) 3a dissolved in dry DMSO. After 24 h stirring at room temperature, 0.3 g (1.86 mmol) dextran was added. The mixture was allowed to react for 24 h at 80 °C under stirring. The product was isolated by precipitation in 0.1M aqueous solution of sodium bicarbonate, filtered and washed several times with water. The product was dried at ambient temperature under vacuum. Other dextran esters (4b-f) 8

Journal Pre-proof were prepared by the same procedure as for Dex40-T-H.

Dex40-T-H (4a) yellow solid, yield: 0.37 g (69%). DS = 58.8 mol % (calculated from N-content determined by elemental analysis). FTIR (KBr)(cm-1): 1735 (νC=O triazole),

1420 (νN=N arom), 1015 (νC-N).

13

ester),

1702 (νC=O

ketone

), 1598 (νC=C

C-NMR (DMSO-d6, 100 MHz) δ (ppm) = 56.1 (C7),

65.4 (C6’), 67.2-76.5 (C2’-C5’), 76.6 (C2’’), 95.3 (C1’’), 98.4 (C1’), 128.2 (C11), 129.8 (C10), 130.8 (C5), 135 (C12), 137.7 (C4), 141.4 (C9), 159.9 (C6), 191.6 (C8).

of

Dex40-T-F (4b)

beige solid, yield: 0.47 g (73%). DS = 80.5 mol % (calculated from N-content determined by 13

triazole

),

C-NMR (DMSO-d6, 100 MHz) δ (ppm) = 56.1

-p

1430 (νN=N arom), 1160 (νC-F), 1018 (νC-N).

ro

elemental analysis). FTIR (KBr)(cm-1): 1734 (νC=O ester), 1703 (νC=O ketone), 1598 (νC=C

(C7), 65.3 (C6’), 67.1-76.4 (C2’-C5’), 76.5 (C2’’), 95.3 (C1’’), 98.2 (C1’), 116.1 (C11), 130.7 (C5),

re

131.4 (C10), 138.5 (C4), 144.3 (C9), 160.1 (C6), 164.7 (C12), 190.4 (C8).

lP

Dex40-T-Cl (4c)

beige solid, yield: 0.45 g (71%). DS = 72.7 mol % (calculated from N-content determined by

na

elemental analysis). FTIR (KBr)(cm-1): 1734 (νC=O ester), 1704 (νC=O ketone), 1588 (νC=C 1432 (νN=N arom), 1093 (νC-Cl), 1014 (νC-N).

C-NMR (DMSO-d6, 100 MHz) δ (ppm) = 56

2’’

ur

2’- 5’

),

(C ), 65.4 (C ), 67-76.3 (C C ), 76.6 (C ), 95.3 (C1’’), 98.2 (C1’), 128.9 (C11), 129.9 (C10), 7

6’

13

triazole

Dex40-T-Br (4d)

Jo

130.8 (C5), 133.4 (C12), 133.8 (C4), 140.6 (C9), 159.9 (C6), 191 (C8).

brown solid, yield: 0.46 g (70%). DS = 66.3 mol % (calculated from N-content determined by elemental analysis). FTIR (KBr)(cm-1): 1732 (νC=O ester), 1704 (νC=O ketone), 1641 (νC-C arom), 1587 (νC=C triazole), 1424 (νN=N arom), 1071 (νC-Br), 1012 (νC-N).

13

C-NMR (DMSO-d6, 100 MHz) δ

(ppm) = 56.4 (C7), 65.4 (C6’), 66-76.4 (C2’-C5’), 76.6 (C2’’), 95.5 (C1’’), 98.4 (C1’), 128.7 (C12), 130.2 (C10), 130.9 (C5), 132.4 (C11), 138.4 (C4), 140.8 (C9), 159.9 (C6), 191 (C8).

Dex40-T-CH3 (4e) beige solid, yield: 0.35 g (67%). DS = 53.6 mol % (calculated from N-content determined by elemental analysis). FTIR (KBr)(cm-1): 1736 cm-1 (νC=O triazole

), 1431 (νN=N arom), 1018 (νC-N).

13

ester),

1697 (νC=O

ketone

), 1543 (νC=C

C-NMR (DMSO-d6, 100 MHz) δ (ppm) = 21.2 (C13), 9

Journal Pre-proof 56.2 (C7), 65.9 (C6’), 67-76.5(C2’-C5’), 76.5 (C2’’), 95.5 (C1’’), 98.2 (C1’), 128.3 (C10), 129.5 (C11), 130.9 (C5), 131.4 (C12), 138.5 (C4), 144.9 (C9), 160 (C6), 191.1 (C8).

Dex40-T-OCH3 (4f) beige solid, yield: 0.51 g (74%). DS = 87.9 mol % (calculated from N-content determined by elemental analysis). FTIR (KBr) (cm-1): 1735 (νC=O ester), 1689 (νC=O ketone), 1574 (νC=C triazole), 1420 (νN=N arom), 1047 (νC(arom)-O-C(aliph)), 1016 (νC-N).13C-NMR (DMSO-d6, 100 MHz) δ (ppm) = 56 (C7), 58.8 (C13), 65.5 (C6’), 67.1-73.3 (C2’-C5’), 76.4 (C2’’), 95.4 (C1’’), 98.1 (C1’), 128.4

of

(C11), 130.1 (C10), 130.8 (C5), 138.4 (C4), 140,8 (C9), 159.1 (C12), 160 (C6), 189.9 (C8).

2.3.1. Preparation of polymeric nanoaggregates

ro

2.3. Methods

-p

The self-aggregates were prepared by a solvent exchange method. Thus, 70 mg of polymer were dissolved in 7 mL of DMSO under magnetic stirring. Then, 7 mL of Millipore water

re

were added drop-wise to DMSO solution under stirring. The mixture was placed into a dialysis bag (12 kDa cut-off) and dialyzed against Millipore water until the conductivity of

lP

the liquid outer the bag was smaller than 5.0 μS cm-1. The aqueous colloidal solution retrieved from the dialysis tube was stable for more than one month. Evaporation to dryness

na

of a known colloidal solution volume afforded the determination of its initial concentration

TEM measurements.

ur

value. The colloidal solution was utilized as such or after dilution for fluorescence, DLS and

1

H- and

13

Jo

2.3.2. Chemical structure characterization C-NMR spectra were obtained in DMSO-d6 with a Bruker Advance DRX 400C

spectrometer (400 MHz for 1H-NMR and 100 MHz for

13

C-NMR), using tetramethylsilane

(TMS) as internal standard. FTIR spectra were recorded with a Bruker Vertex 70 spectrophotometer on KBr pellets. Elemental analysis (N%) was carried out by means of CHNS 2400 II Perkin Elmer analyzer. 2.3.3. Thermal analysis Thermal decomposition of the products was studied with Perkin Elmer Pyris Diamond thermogravimetric analyzer. A heating rate of 10 °C/min was kept constant throughout the analysis along with temperatures ranging between room temperature to 600 °C. Nitrogen was used as purge gas for keeping inert atmosphere for the samples. Differential scanning 10

Journal Pre-proof calorimetry (DSC) was carried out on a Perkin-Elmer Pyris Diamond DSC (Perkin-Elmer Instruments, USA) at a heating/cooling rate of 10 °C/min in the range of -40° to 450 °C under a steady flow of nitrogen.

2.3.4. Wide-angle X-ray diffraction (WAXD) technique The X-ray power diffraction data were performed with a Bruker AD8 Advance difractometer with Cu K radiation at 40 kV and 200 mA, at room temperature. Scattered radiation was detected in the diffraction angle 2 ranging from 10 to 40 at a rate of 2 min-1.

of

2.3.5. Fluorescence measurements

ro

The critical aggregation concentration (CAC) of polymer micelles were established using pyrene as extrinsic fluorescence probe. Polymer concentrations, varying from 0.66 g/dL to

-p

0.000177 g/dL, were made by consecutive dilution of polymeric aggregates in aqueous solution obtained according to the preparation of polymeric nanoaggregates described in

re

section 2.3.1. A known amount of pyrene solution (5.10-4M) in methanol was added to a

lP

series of vials, followed by solvent vaporation. After that, polymer solutions of known concentrations were put into each vial and all the mixtures were kept at room temperature for 24 h to ensure that pyrene was completely entrapped into the polymers’ hydrophobic

na

microdomains. The final concentration of pyrene in vials was 5.0 .10-7 mol/L. Pyrene was excited at 337 nm and the excitation and emission slits widths were set at 5 and 2 nm,

ur

respectively. The emission spectra of pyrene were recorded between 350–550 nm using a LS

Jo

55 PerkinElmer fluorescence spectrometer. The emission intensities, registered at 372 nm (I1) and 383 nm (I3), were utilized for the calculation of the ratio I1/I3 (polarity parameter). 2.3.6. Light scattering measurements The average particle size and size distribution of the amphiphilic polymers were measured by dynamic light scattering (DLS) using a Zetasizer model Nano ZS, with red laser 633 nm He/Ne (Malvern Instruments, UK) on 50 mg/dL esters aqueous solution achieved as reported in section 2.3.1. The measurements were performed in duplicate for each sample. The size of particles was evaluated using the technique of cumulants. Self-aggregates size was assessed by using intensity distribution.

2.3.7. Transmission electron microscopy

11

Journal Pre-proof Transmission electron microscopy (TEM) was recorded using a HITACHI T7700 microscope (Tokyo, Japan), operating at an acceleration voltage of 120 kV in high resolution mode. For evaluating the morphology of aggregates, a drop of polymeric micelles, obtained in aqueous solution in agreement with the preparation of polymeric nanoaggregates described in section 2.3.1., was placed onto a copper grid coated with carbon, then was negatively stained with 2% (w/v) phosphotungstic acid (PTA) aqueous solution and air dried at room temperature.

2.3.8. Antimicrobial activity The antimicrobial activity was studied using Gram positive bacteria (Staphylococcus aureus

of

ATCC 25923), Gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas

ro

aeruginosa ATCC 27853) and pathogenic yeasts (Candida albicans ATCC 90028 and Candida albicans ATCC 14053). The antimicrobial activity was evaluated using the disk

-p

diffusion methods [38, 39]. Mueller Hinton agar (Oxoid) and Mueller-Hinton agar Fungi (Biolab) were inoculated with the suspensions of the tested microorganisms. Sterile stainless

re

steel cylinders (5 mm internal diameter; 10 mm height) were applied on the agar surface in Petri plates. Then, 100 µL of each tested compounds (4a, 4e and 4f, as 1 wt% DMSO

lP

solutions) were added into cylinders. The plates were left 10 minutes at room temperature to ensure the equal diffusion of the compound in the medium and then incubated at 35ºC for 24

na

hrs. Commercial available discs containing Ciprofloxacin (5 µg/disk), Fluconazole (25 μg/disk) and Voriconazole (1 μg/disk) were used as reference antimicrobial drugs. After

Jo

ur

incubation, the diameters of inhibition were measured in mm, including disc size.

3. Results and discussion

3.1. Synthesis and chemical structure characterization The synthetic route for dextran esters is presented in Scheme 1. The reaction between organic azides 1a-d and ethyl propiolate in the presence of copper(II) sulfate pentahydrate and sodium L-ascorbate was successful. The formation of esters 2a-f by CuAAC click reaction was confirmed by FTIR analysis as well as by NMR spectroscopy (1H - and

13

C). Thus, the peaks assigned to the protons of C5, Ca and Cb, appeared in 1H-NMR

spectra of esters at 8.70-8.72 ppm, 4.31-4.34 ppm and 1.31-1.33 ppm, respectively. 13C-NMR spectra proved the formation of 2a-f by the occurrence of the peaks, attributed to C5, Ca and Cb, at 130.5-131.5 ppm, 60.4-61.3 ppm and 14.0-14.9 ppm, respectively. FTIR spectra of 2a-f showed significant signals at 1720-1726 cm-1 typical for the ester moiety, 1434-1475 cm-1 12

Journal Pre-proof and 1564-1611 cm-1 attributed to C=Ctriazole. 1,2,3-triazole-

characteristic for N=Narom

carboxylic acids 3a-f were obtained by the hydrolysis of ethyl 1H-1,2,3-triazole-4carboxylates 2a–f in 30 % KOH aqueous solution. FTIR studies and NMR (1H- and

13

C)

1

spectroscopy confirmed the formation of 3a–f. Thus, H-NMR spectra showed that the peaks, assigned to the protons of Ca and Cb, disappeared and a new peak at 13.00-13.25 ppm, attributed to the proton of C6, appeared. Also,

13

C-NMR spectra of products 3a-f revealed

that the peaks for Ca and Cb were missing and a new peak at 160.1-161.1 ppm, attributed to C6, appeared. In FTIR spectra of the carboxylic acids 3a-f a new signal at 1676-1698 cm-1, attributed to C=Oacid, was found while the signal for C=Oester disappeared.

of

The polysaccharide esters were obtained using an one-pot reaction. Thus, 3a-f reacted with

ro

CDI to give the activated product (acylimidazole) and two by-products, CO2 and imidazole (Im). Then, acylimidazolide reacted in situ with dextran to give polysaccharide esters. The

-p

structural characterization of the polymers was carried out by elemental analysis, FTIR and 13

C NMR spectroscopy. Elemental analysis was performed in order to confirm the successful

re

attachment of 1,2,3-triazoles on the polysaccharide and allow to find DS values. The degree of substitution (Table 1), expressed in moles of N-heterocycle group/100 glucopyranosidic 162 x %N *100, mole/100 UGl 100 x 3×14 - %N x (MS−1)

na

DS =

lP

units (UGl), was calculated with the equation (1).

(1)

ur

% N is the nitrogen percent, determined by elemental analysis, MS is the molecular weight of the polymeric pendant group (-CO-1,2,3-Tr-CH2-CO-C6H4-A)

Jo

(Tr = 1,2,3-triazole).

Table 1

Polysaccharide derivatives were obtained by nucleophilic acyl substitution between the nucleophile (dextran) and acyl compound (acylimidazolide) which was obtained in situ. In the first stage of the nucleophilic substitution, dextran attacked the carbonyl group of the acyl compound to give a tetrahedral alkoxide intermediate which expelled, in the second stage, the leaving group (-Im) to give the substitution product. DS values were moderate due to the steric hindrance exerted by the two bulky groups (-1,2,3-Tr-CH2-CO-C6H4-A and –Im) bound, in the first stage of nucleophilic substitution, to the carbonyl group of acylimidazolide. DS values decreased with the decrease of 1,4-disubstituted triazoles solubility in the reaction medium [40] which required an increased dilution of the medium. So, the degree of substitution decreased in the following order: DSDex-T-OCH3 > DSDex-T-F > 13

Journal Pre-proof DSDex-T-Cl > DSDex-T-Br > DSDex-T-H > DSDex-T-CH3. Polysaccharide derivatives were also analyzed by FTIR and

13

C NMR. The formation of esters was confirmed by FTIR

spectroscopy which yielded a signal in the range 1732-1736 cm-1, attributed to the carbonyl group of the ester. The products were analyzed by

13

C-NMR spectroscopy due to a limited

separation of the peaks for dextran-based polymers in the 1H NMR spectra. The

13

C NMR

spectrum of Dex40-T-CH3 was presented as an example (Fig.1). Fig.1 The signals corresponding to first carbon atom of dextran glucopiranosidic ring could provide information about substitution at second carbon (C2’). Thus, the first carbon adjacent to

of

unsubstituted second carbon, namely C1’, could be seen at 98.27 ppm while the first carbon adjacent to substituted second carbon (C1’’) could be found at 95.37 ppm. The presence of

ro

both signals at 95.37 and 98.27 ppm indicated an incomplete substitution in C2’. The pick

-p

attributed to substituted second carbon, namely C2’’, could be seen at 76.6 ppm. The signals corresponding to substituted C3’ or C4’ could not be found, pointing out that the substitution

re

occurred preferentially at C2’–OH, which is the most reactive hydroxyl group of dextran.

lP

Carbon atoms resonances assigned to the 1,4-disubstituted 1,2,3-triazole moiety were determined at 21.2, 56.2, 128.3, 129.5, 130.9, 131.4, 138.5, 144.9, 160.0 and 191.1 ppm for

3.2. Thermal properties

na

C13, C7, C10, C11, C5, C12, C4, C9, C6 and C8, respectively.

ur

3.2.1. Thermogravimetric studies

Jo

Thermal stability of dextran derivatives was studied by thermogravimetric analysis. During the early stage of native polysaccharide degradation there is a sudden drop in the thermogravimetric curve. This may be assigned to the elimination of bound water from dextran. 50% weight loss for dextran was record at 309 °C. The result was in good agreement with the value reported in the literature for the thermal decomposition of this polysaccharide [41]. Dextran esters revealed a single stage degradation, as their water content was low due to their hydrophobic nature. 50% weight loss was reached at 350.2, 359.4, 367.1, 375, 379 and 381.6 °C for 4e, 4a, 4d, 4c, 4f, 4b, respectively (Fig. 2). Although the existence of different substituents in the para position of the aromatic ring made difficult a direct comparison of polymers’ thermal stability, an increase of T50 values was clearly observed with the increase of DS (insert in Fig.2). The improve of thermal stability after esterification could also be seen for other polysaccharides, such as: pullulan [42], starch [43], xylan [44,45], hemicellulose

14

Journal Pre-proof [46] and glucomannan [47, 48]. Fig. 2

3.2.2. Differential scanning calorimetry analysis The thermal transitions for polymers were studied by differential scanning calorimetry. The measurements were performed with two consecutive heating–cooling cycles. The first heating process was employed to erase thermal history, and the second heating run was registered for data analysis. The second DSC heating curves (Fig. 3) were examined in the temperature range of -10°C to 120°C and the obtained curves pointed out only glass

of

transitions for all esters. Fig. 3

ro

Native dextran, having Mr = 40 KDa, showed a glass transition value at 220 °C (data not

-p

shown). This value is similar with Tg value reported in the literature for this polysaccharide [49]. Glass transition values for the esters ranged from 73.2 to 83.5 °C. Tg decreased due to

re

the chemical modification of the polysaccharide which resulted in a reduction of the number of hydroxyl groups accessible for the formation of hydrogen bonds and the appearance of

lP

ester bonds, both effects determing an increase of the polymer flexibility. Glass transition values decreased mainly with increasing DS, i.e: Tg(Dex-40-T- CH3) > Tg(Dex-40-T- H) > Tg(Dex40-T- Br)

na

> Tg(Dex40-T -Cl ) > Tg(Dex40-T- OCH3) > Tg(Dex40-T- F)(insert in Fig.3). The same variation of Tg with

ur

DS was also observed for other polysaccharide esters [42, 47, 48, 50, 51].

Jo

3.3. Wide-angle X-ray diffraction studies Dextran presents a broad peak centered at 2Φ=180, suggesting some level of crystallinity [52]. In addition to the crystalline region, dextran also has an amorphous area (Fig. 4). The existence of inter- and intramolecular hydrogen bonds was the main cause for dextran crystallinity [53,54]. WAXD spectra of all dextran-based polymers showed a more broad peak at 2Φ = 18° than that showed by native polysaccharide. This peak decreased mostly with increasing DS values of esters (Fig. 4). The amorphous structure of the polymers, indicated by WAXD studies, was confirmed by the absence of any melting point detected in DSC analysis. Fig. 4 The introduction of 1,2,3-triazole derivatives onto dextran backbone decreased the amount of free hydroxyl groups, thus weakening the inter- and intramolecular hydrogen bonds [53,54]. 15

Journal Pre-proof The reduction of the crystallinity after esterification could be seen for various polysaccharides, such as: cellulose [55,56], xylan [44], pullulan [42], starch [57], etc.

3.4. Self-organization of dextran esters in aqueous solutions The main driving force in self-association of amphiphilic polymers is the loss of entropy due to the disruption of hydrogen bonds between water molecules that surround the hydrophobic moieties of the polymers. As a consequence, the non-polar segments tend to be expelled from the aqueous media and are screened by the hydrophilic fragments causing the formation of the hydrophobic microdomains.

of

Dex40-T-A could self-assemble in aqueous media and their self-aggregates were prepared by

ro

a solvent-exchange method. The capacity to self-associate in aqueous media was studied by

-p

fluorescence and aggregate properties were established by DLS and TEM analysis.

3.4.1. Critical aggregation concentration of polymeric self-aggregates

re

The aggregation capacity of polysaccharide esters in the aqueous media was examined by fluorescence spectroscopy using pyrene as free chromophore. The variation in the ratio of

lP

intensity of first (372 nm) to the third (383 nm) vibronic peaks (I1/I3), the polarity parameter, is fairly sensitive to the polarity of hydrophobic microdomains where the extrinsic fluorescent

na

probe is placed [58,59]. Fig. 5 showed the variation of pyrene polarity parameter with Dex40-T-H concentration. At low polymer concentrations, the values of I1/I3 remained nearly

ur

unchanged. Furthermore, increase of polymer concentration determined the decrease of the polarity parameter, due to the self-assembly capacity of dextran-based polymers. CAC for

Jo

Dex40-T-H was established to be 9.5 mg/dL by the interception of two straight lines. Fig. 5

Critical aggregation concentration values correlated better with hydrophobic groups content (wt%) and decreased with increasing this content (Table 1). Compared to low molecular weight surfactants, the polysaccharide esters had a lower CAC value. This suggests an easier formation and also a better colloidal stability meaning a better stability to dilution in comparison with those of low molecular weight surfactants. Dextran-based polymers with the same molar mass of the polysaccharide (40 KDa) and having as side chains, lipophilic moieties with different chemical structure and content (wt%) make difficult a direct comparison between their critical aggregation concentration values. However, CAC values showed by Dex40-T-A, having hydrophobic weight content in the range of 35.1-43 wt %, are consistent with the values reported for other dextran amphiphilic polymers with 8.7 wt% 16

Journal Pre-proof tocopherol succinate (0.34 mg/dL) [60], 11.2 wt% stearic acid (1 mg/dL) [61], 9.6 wt% retinal (2.5 mg/dL) [62].

3.4.2. Size and morphology of the polymeric self-associates The size of polymeric aggregates and their size distribution in aqueous medium were assessed by dynamic light scattering. A unimodal distribution for polymers self-aggregates (Fig. 6) could be observed. Fig. 6 The values for the mean diameter (d)(Table 1) decreased with increasing hydrophobic

of

content (wt%), demonstrating the formation of more compact hydrophobic cores. TEM

Fig. 7

ro

measurement showed isolated and spherical particles with a unimodal distribution (Fig. 7).

-p

The size of the micelles determined by TEM (185-225 nm) was smaller than that determined by DLS (263-295 nm) due to the dried state of the polymeric self-aggregates required by

re

TEM measurement while DLS method involved the measurement of the size in the hydrated state of the samples [63]. Other amphiphilic dextran esters, having the same molar mass of

lP

the polysaccharide (40 KDa), formed self-aggregates in the aqueous medium with similar [60] or smaller [61,62,64] diameter values than those reported in this work, depending on the

na

chemical structure of the pendant groups and DS values. The larger size of Dex-T-A micelles can be explained by the less closely packed structure of polymer chains as a result of the

ur

structural planarity of 1,2,3-triazole and phenyl rings, respectively [65-67].

Jo

4. Antimicrobial activity

Preliminary studies on antimicrobial activity of Dex-T-A (A=H, CH3, OCH3) were carried out using disc-diffusion method. The diameters of the inhibition zones (in mm) corresponding to the tested compounds are shown in Table 2. All tests were performed in triplicate and the results are expressed as mean diameter ± standard deviation (SD). Table 2 The antimicrobial analysis confirmed that the presence of 1,4-disubstituted 1,2,3-triazole groups, as side-chains of dextran-based polymers, induced antimicrobial activity for tested polymers. It is known that triazole ring is effective antimicrobial functional group because it inhibits synthesis of the cell membrane and cell wall [68]. It was expected that the presence of -CH3 and -OCH3, like substituent groups on para position of aromatic ring, enhanced the antimicrobial activity as was reported in the literature [69]. The results pointed out that Dex17

Journal Pre-proof T-CH3 and Dex-T-OCH3 showed a good antimicrobial activity against S. aureus ATCC 25923 while Dex-T-H had a moderate antimicrobial activity. Between tested polymers only Dex-T-OCH3 proved to have a moderate antimicrobial activity against E. coli ATCC 25922 and P. aeruginosa ATCC 27853 whilst Dex-T-H and Dex-T-CH3 didn’t have any effect. A good antifungal activity against C. albicans ATCC 90028 and C. Albicans ATCC 14053 was observed for Dex-T-H whereas Dex-T-OCH3 and Dex-T-CH3 revealed a moderate antifungal activity. Dependence of antimicrobial activity on type of microbes, examined by broth microdilution method (minimum inhibitory concentrations), and the study of structure– activity relationship between the antimicrobial activities and the physicochemical /structural

ro

of

properties for all dextran esters will be given in a forthcoming paper.

5. Conclusions

-p

New dextran-based amphiphilic esters were prepared by coupling different substituted 1,2,3triazole-4-carboxylic acids to the polysaccharide. 1,2,3-triazole derivatives were achieved by

re

copper-catalyzed azide-alkyne cycloaddition. The polymers showed good thermal stability and amorphous structure. Hydrophobically modified polymers were able to self-assemble in

lP

aqueous medium into stable spherical micelles with a unimodal distribution. Critical aggregation concentration for the polysaccharide esters demonstrated lower values when

na

compared with those of low molecular weight surfactants. Some of the polymers, tested against Gram positive or Gram-negative bacteria and fungi strains, proved to have a moderate

Jo

Acknowledgements

ur

to good antimicrobial activity.

This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN-III-P4-ID-PCE-2016-0519. Dalila

Belei

thanked

the

POSCCE-O

2.2.1,

no.257/28.09.2010, CERNESIM, for the NMR analysis.

References

18

SMIS-CSNR

13984-901,

project

Journal Pre-proof

[1] J.N. BeMiller, Dextran, in: B. Caballero, L. Trugo, P.M. Finglas (eds), Encyclopedia of food Sciences and Nutrition (2-nd edn.) Elsevier Science BV, Amsterdam, 2003, pp 1772– 1773. [2] H.A. Staab, Neuere Methoden der präparativen organischen Chemie IV Synthesen mit heterocyclischen Amiden (Azoliden), Angew. Chem. 74 (1962) 407-423. https://doi.org/ 10.1002/ange.19 620741203. [3] A.S. Williams, G. Taylor, Synthesis, characterization and release of cromoglycate from

of

dextran conjugates, Int. J. Pharm. 83 (1992) 233-239. https://doi.org/10.1016/03785173(82)90027-8.

ro

[4] E. Harboe, C, Larsen, M. Johansen, H.P. Olesen, Macromolecular prodrugs. XV. Colon-

-p

targeted delivery-bioavailability of naproxen from orally administered dextran-naproxen ester

doi.org/10.1023/ A:1015981126732.

re

prodrugs varying in molecular size in the pig, Pharm. Res. 6 (1989) 919-923. https://

[5] S. Hornig, H. Bunjes, Th. Heinze, Preparation and characterization of nanoparticles based

lP

on dextran–drug conjugates, J. Colloid. Interface. Sci. 338 (2009) 56–62. https://doi.org /10.1016/j.jcis. 2009.05.025.

na

[6] F. Levi-Schaffer, A. Bernstein, A. Meshorer, R. Arnon, Reduced toxicity of daunorubicin by conjugation to dextran, Cancer Treat. Rep. 66 (1982) 107-114.

ur

[7] T. Kojima, M. Hashida, S. Muranishi, J. Sezaki, Mitomycin C-dextran conjugate: a novel high molecular weight pro-drug of mitomycin C., J. Pharm. Pharmacol. 32 (1980) 30-34.

Jo

https://doi. org/10.1111/j.2042-7158.1980. [8] Y. Ohya, H. Oue, K. Nagatomi, T. Ouchi, Design of macromolecular prodrug of cisplatin using dextran with branched galactose units as targeting moieties to hepatoma cells, Biomacromolecules 2 (2001) 927-933. https://doi.org/10.1021/bm010053o. [9] C. Larsen, B.H. Jensen, Bioavailability of ketoprofen from orally administered ketoprofen-dextran ester prodrugs in the pig, Acta Pharm. Nord 3 (1991) 371-376. [10] P.K. Shrivastava, A. Shrivastava, S.K. Sinha,

S.K. Shrivastava, Dextran Carrier

Macromolecules for Colon-specific Delivery of 5-Aminosalicylic Acid, Indian J. Pharm. Sci. 75 (2013) 277–283. https://doi.org/10.4103/0250-474X.117420. [11] P.K. Shrivastava, S.K. Shrivastava, Dextran carrier macromolecule for colon specific delivery of celecoxib, Curr. Drug Deliv. 7 (2010) 144-151. https://doi.org /10.2174 /156720110791011 828. 19

Journal Pre-proof

[12] J.S. Lee, Y.J. Jung, M.J. Doh, Y.M. Kim, Synthesis and properties of dextran-nalidixic acid ester as a colon-specific prodrug of nalidixic acid, Drug Dev. Ind. Pharm. 27 (2001) 331-336. https://doi.org/10.1081/DDC-100103732. [13] H. Wondraczek, Th. Heinze, Efficient Synthesis and Characterization of New Photoactive Dextran Esters Showing Nanosphere Formation, Macromol. Biosci. 8 (2008) 606–614. https://doi.org/10.1002/mabi.200800056. [14] C. Grote, Th. Heinze, Starch Derivatives of High Degree of Functionalization 11: Studies on Alternative Acylation of Starch with Long-chain Fatty Acids Homogeneously in

of

N,N-dimethyl acetamide/LiCl, Cellulose 12 (2005) 435-444. https://doi.org/10.1007/s10570005-2178-z.

ro

[15] J. Han, G. Borjihan, R. Bai, Zh. Sun, N. Bao, X. Chen, X. Jing, Synthesis and anti-

-p

hyperlipidemic activity of a novel starch piperinic ester, Carbohydr. Polym. 71 (2008) 441– 447. https:// doi.org/10.1016/j.carbpol.2007.06.014.

re

[16] T.F. Liebert, Th. Heinze, Tailored cellulose esters: synthesis and structure determination, Biomacromolecules 6 (2005) 333-340. https://doi.org/10.1021/bm049532o.

lP

[17] D. Gräbner, T. Liebert, Th. Heinze, Synthesis of Novel Adamantoyl Cellulose Using Differently Activated Carboxylic Acid Derivatives, Cellulose 9 (2002) 193-201. https://

na

doi.org /10.1023/A:1020120427308

[18] M.A. Hussain, K. Abbas, B.A. Lodhi, M. Sher, M. Ali, M.N. Tahir, W. Tremel, S. Iqbal

ur

Fabrication, characterization, thermal stability and nanoassemblies of novel pullulan-aspirin

06.001.

Jo

conjugates, Arab J. Chem. 10 (2017) S1597-S1603. https://doi.org/10.1016/j.arabjc. 2013.

[19] M.A. Hussain, Th. Heinze, Unconventional synthesis of pullulan abietates, Polym. Bull. 60 (2008) 775–783. https://doi.org/10.1007/s00289-008-0918-6 [20] H.C. Kolb, M.G. Finn, K.B. Sharpless, Click Chemistry: Diverse Chemical Function from a Few Good Reactions, Angew. Chem. Int. Ed., 40(11) (2001) 2004–2021. https:// doi.org/ 10.1002 /1521-3773(20010601)40:11. [21] S. Danoun, G. Baziard-Mouysset, J. Stigliani, M. Payard, M. Selkti, B. Viossat, A. Tomas, Addition of diazomethane to 3 and 4-nitrophthalodinitriles, Heterocycl. Commun. 4 (1998) 45–51. https://doi.org/10.1515/HC.1998.4.1.45. [22] A. Passannanti, P. Diana, P. Barraja, F. Mingoia, A. Lauria, G. Cirrincione, Pyrrolo[2,3d][1,2,3]triazoles as Potential Antineoplastic Agents, Heterocycles 48 (1998) 1229–1235. https://doi.org/10.1002/chin.199839130. 20

Journal Pre-proof

[23] M.D. Chen, S.J. Lu, G.P. Yuag, S.Y. Yang, X.L. Du Synthesis and Antibacterial Activity of some Heterocyclic β-Enamino Ester Derivatives with 1,2,3-triazole, Heterocyclic Comm. 6 (2000) 421–426. https://doi.org/10.1515/HC.2000.6.5.421 [24] E.A. Sherement, R.I .Tomanov, E.V. Trukhin, V.M. Berestovitskaya, Synthesis of 4Aryl-5-nitro-1,2,3-triazoles, Russ. J. Org. Chem. 40 (2004) 594–595. https://doi.org/10.1023/ B:RUJO. 0000036090.61432.18. [25] M. Jilino, M.F.G. Stevens, Antitumour polycyclic acridines. Part 5.1 Synthesis of 7Hpyrido[4,3,2-kl]acridines with exploitable functionality in the pyridine ring, J. Chem. Soc.

of

Perkin Trans 1 10(10) (1998) 11677–1684. https://doi.org/10.1039/a800575c. [26] J.D. Shukla, A. K. Arif, K. Deo, Synthesis and pharmacological evaluation of novel 1-

ro

(2, 6-difluorobenzyl)-1H-1,2,3-triazole derivatives for CNS depressant and anticonvulsant profile, Am. J. Pharm. Tech. Res. 5 (2015) 423–433.

-p

[27] Y.W. He, C.Z. Dong, J.Y. Zhao, L. Ma, Y.H. Li, H.A. Aisa, 1,2,3-Triazole-containing

re

derivatives of rupestonic acid: Click-chemical synthesis and antiviral activities against influenza viruses, Eur. J. Med. Chem. 76 (2014) 245-255. https://doi.org/10.1016/

lP

j.ejmech.2014.02.029.

[28] G. Sadaf, K. Shakila, A. Ozair, K. Harish, Synthesis and biological evaluation of some

na

substituted -1,2,3-triazole derivatives, Indian J. Heterocycl. Chem. 17 (2008) 245-248. [29] M. Whiting, J.Muldoon, Y.C. Lin, M.S. Silverman, W. Lindstron, A.J. Olson, H.C.

ur

Kolb, M.G. Finn, K.B. Sharpless, J.H. Elder, V.V. Fokin, Inhibitors of HIV-1 protease by using in situ click chemistry, Angew. Chem. Int. Ed. 45 (2006) 1435–1439. https://doi.org

Jo

/10.1002/ anie.200502161.

[30] G.R. Labadie, A. Iglesia, H.R. Morbidoni, Targetting tuberculosis through a small focused library of 1,2,3-triazoles, Mol. Divers. 15 (2011) 1017–1024. ttps://doi.org/10.1007/ s11030-011- 9319-0. [31] Q. Ma, S. Qi, X. He, Y. Tang, G. Lu, 1,2,3-Triazole derivatives as corrosion inhibitors for mild steel in acidic medium: Experimental and computational chemistry studies, Corros. Sci. 129 (2017) 91–101. https://doi.org/10.1016/j.corsci.2017.09.025. [32] L. Marin, S. Shova, C. Dumea, E. Bicu, D. Belei, Self-assembled Triazole AIE-Active Nanofibers: Synthesis, Morphology, and Photophysical Properties, Cryst. Growth & Des. 17(7) (2017) 3731–3742. https://doi.org/10.1021/acs.cgd.7b00351. [33] D. Belei, C. Dumea, E. Bicu, L. Marin, Phenothiazine and pyridine-N-oxide-based AIEactive triazoles: synthesis, morphology and photophysical properties, RSC Advances 5(12) 21

Journal Pre-proof

(2015) 8849–8858. https://doi.org/10.1039/C4RA13383H. [34] N.G. Aher, V.S. Pore, N.N. Mishra, A. Kumar, P.K. Shukla, A. Sharma, M.K. Bhat, Synthesis and antifungal activity of 1,2,3-triazole containing fluconazole analogues, Bioorg. Med. Chem. Lett. 19(3) (2009) 759–763. https://doi.org/10.1016/j.bmcl.2008.12.026. [35] D.G. Luster, P.A. Miller, Triazole Plant Growth Regulator Binding to Native and Detergent-Solubilized Plant Microsomal Cytochrome P450, Pestic. Biochem. Physiol. 46(1) (1993) 27–39. https://doi.org/10.1006/pest.1993.1033. [36] L. Lucescu, E. Bicu, D. Belei, S. Shova , B. Rigo, Ph. Gautret, J. Dubois, A. Ghinet,

of

human

farnesyltransferase,

Res.

Chem.

Intermed.

42(3)

(2016)

1999–2021.

ro

https://doi.org/10.1007/s11 164 -015-2131-1.

of

Synthesis and biological evaluation of a new class of triazin–triazoles as potential inhibitors

[37] Dumea C., Synthesis and biological evaluation of novel pentaatomic nitrogen-containing

-p

heterocyclic derivatives, PhD Thesis (2014) University of Iasi, Romania.

re

[38] CLSI, Method for antifungal disk diffusion susceptibility testing of yeasts; approved guideline (2nd ed.). P.A. Wayne, Clinical and Laboratory Standards Institute, CLSI document

lP

M44-A2 (2009).

[39] CLSI, Performance standards for antimicrobial susceptibility testing (26th ed.). P.A.

na

Wayne, Clinical and Laboratory Standards Institute, CLSI supplement M100S (2016). [40] C. Reichardt, Solvatochromic Dyes as Solvent Polarity Indicators, Chem. Rev. 94 (1994)

ur

2319-2358. https://doi.org/10.1021/cr00032a005. [41] M. Amin, M.A. Hussain, D. Shahwar, M. Hussain, Thermal analysis and degradation

673-679.

Jo

kinetics of dextran and highly substituted dextran acetates, J. Chem. Soc. Pak. 37 (2015)

[42] Y. Enomoto-Rogers, N. Iio., A.Takemura, T. Iwata, Synthesis and characterization of Pullulan Alkyl Esters, Eur. Polym. J. 66 (2015) 470–477. https://doi.org/10.1016/j.eurpolymj .2015.03. 007. [43] J. Aburto, S. Thiebaud, I. Alric, E. Borredon, D. Bikiaris,

J. Prinos, C.

Panayiotou, Synthesis, characterization and biodegradability of fatty-acid esters of amylose and starch, J. Appl. Polym. Sci. 74 (1999)1440-1451. https://doi.org/10.1002/(SICI)10974628(19991107)74:6<1440::AID-APP17>3.0.CO;2-V. [44] N.G.V. Fundador, Y. Enomoto-Rogers, A. Takemura, T. Iwata, Syntheses and characterization of xylan esters, Polymer 53(18) (2012) 3885–3893. https://doi.org /10.1016/j.polymer.2012.06.038. 22

Journal Pre-proof

[45] P. Skalková, K. Csomorová, Preparation of 4-O-methylglucuronoxylan cinnamates, Procedia Eng. 136 (2016) 328 – 335. https://doi.org/10.1016/j.proeng.2016.01.21. [46] F. Peng, J.L. Ren, B. Peng, F. Xu, R.C. Sun, J.X. Sun, Rapid homogeneous lauroylation of wheat straw hemicelluloses under mild conditions, Carbohydr. Res. 343 (2008) 2956– 2962. https://doi.org/10.1016/j.carres.2008.08.023. [47] Y. Enomoto-Rogers, Y. Ohmomo, T. Iwata, Syntheses and characterization of konjac glucomannan acetate and their thermal and mechanical properties, Carbohydr. Polym. 92(2) (2013) 1827–1834. https://doi.org/10.1016/j.carbpol.2012.11.043.

of

[48] T. Danjo, Y. Enomoto-Rogers, A. Takemura, T. Iwata, Syntheses and properties of glucomannan acetate butyrate mixed esters, Polym. Degrad. Stab. 109 (2014) 373–378.

ro

https://doi.org/10.1016/j.polymdegradstab.2014.05.023.

[49] M.G. Cascone, G. Polacco, L. Lazzeri, N. Barbani, Dextran/Poly (acrylic acid) Mixtures

-p

as Miscible Blends, J. Appl. Polym. Sci. 66 (1997) 2089–2094. https://doi.org/10.1002/

re

(SICI)1097-4628 (19971219) 66.

[50] Y. Matsumoto, D. Ishii, T. Iwata, Synthesis and characterization of alginic acid ester

lP

derivatives, Carbohydr. Polym. 171(2017) 229–235. https://doi.org/10.1016/j.carbpol. 2017.05.001

na

[51] Y. Teramoto, Functional Thermoplastic Materials from Derivatives of Cellulose and Related Structural Polysaccharides, Molecules 20 (2015) 5487-5527. https://doi.org

ur

/10.3390/molecules 20045487.

52. W. Yuan, Y. Geng, F. Wu, Y. Liu, M. Guo, H. Zhao, T. Jin, Preparation of

Jo

polysaccharides glassy microparticles with stabilization of proteins, Int. J. Pharm. 366 (2009) 154- 159. https://doi.org/10.1016/j.ijpharm.2008.09.007. [53] C. Shuang, W. Hao, H. Jing-han, Y. Jing-wen, Zh. Hong-bin, H. Xue-qin, The effect of NaOH and NaClO/NaBr modification on the structural and physicochemical properties of dextran, New J. Chem. 42 (2018) 6274-6282. https://doi.org/10.1039/C7NJ04341D. [54]. J.P. Wang, S.J. Yuan, Y. Wang, H.Q. Yu, Synthesis, characterization and application of a novel starch-based flocculant with high flocculation and dewatering properties, Water Res. 47 (8) (2013) 2643-2648. https://doi.org/10.1016/j.watres.2013.01.050. [55] G. Gong, X. Lu, F. Zhang, Preparation and Characterization of Nanocellulose Grafted Epoxy Groups, Polym. Mat. Sci. Eng. 34(9) (2014) 58-62. https://doi.org/10.1007/s13233014-2121-y. [56] E. Sîrbu, S. Eyley, W. Thielemans, Coumarin and carbazole fluorescently modified 23

Journal Pre-proof

cellulose nanocrystals using a one-step esterification procedure, Can. J. Chem. Eng. 94(11) (2016) 2186-2194 . https://doi.org/10.1002/cjce.22624. [57] J. Aburto, H. Hamaili, M.B. Geneviève, I. Alric, E. Borredon , Free-solvent synthesis and properties of higher fatty esters of starch - Part 2, Starch/Staerke, 51(8-9)(1999) 302-307. https://doi.org/10.1002/(sici)1521-379x(199909)51:8/9<302::aid-star302>3.0.co;2-e. [58] K. Kalyanasundaram, J.K. Thomas, Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems, J. Am. Chem. Soc. 99 (1977) 2039-2044. https://doi.org/10.1021/ja00449a004.

of

[59] F.M. Winnik, S.T.A. Regismond, Fluorescence methods in the study of the interactions

https://doi.org /10.1016/0927-7757(96)03733-8.

ro

of surfactants with polymers, Colloids Surf. A: Physicochem. Eng. Asp. 118 (1996) 1-39.

[60] Y. Jingmou, Zh. Yunfeng, Ch. Wencong, R. Jin, Zh. Lifang, L. Lu, L. Gan, H. Hao,

-p

Preparation, Characterization and Evaluation of α-Tocopherol Succinate-Modified Dextran

re

Micelles as Potential Drug Carriers, Materials 8 (2015) 6685–6696. https://doi.org/ 10.3390/ma8105332.

lP

[61] D. Yong-Zhong, W. Qi, Y. Hong, H. Fu-Qiang, Synthesis and Antitumor Activity of Stearate-g-dextran Micelles for Intracellular Doxorubicin Delivery, ACS Nano 4 (2010)

na

6894-6902. https://doi.org/10.1021/nn100927t.

[62] Zh. Yijuan, L.Ping, P. Hong, L. Lanlan, J. Manyi, S. Nan, W. Ce, C. Lintao, M. Yifan,

ur

Retinal-conjugated pH-sensitive micelles induce tumor senescence for boosting breast cancer

2016.01.023.

Jo

chemotherapy, Biomaterials 83 (2016) 219-232. https://doi.org/10.1016/j.biomaterials.

[63] M.C. Stanciu, M. Nichifor, New biocompatible amphiphilic diblock copolymer based on dextran, Eur. Polym. J. 71 (2015) 352–363. https://doi.org/10.1016/j.eurpolymj.2015.08.011. [64] L. Peng, Y. Caixia, S. Zonghai, G. Guanhui, L. Mingxing, Y. Huqiang, Z. Cuifang, W. Bi, C. Lintao, Photosensitizer-conjugated redox-responsive dextran theranostic nanoparticles for near-infrared cancer imaging and photodynamic therapy, Polym. Chem. 5 (2014) 874881. https://doi.org/ 10.1039/C3PY01173A. [65] H. Gallardo, A.J. Bortoluzzi, D.M. Pereira De Oliveira Santos, Synthesis, crystalline structure and mesomorphic properties of new liquid crystalline 1,2,3-triazole derivatives, Liq. Cryst. 35 (2008) 719–725. https://doi.org/ 10.1080/02678290802120307. [66] M.S. Costa, N. Boechat, E.A. Rangel, F.C. da Silva, A.M.T. de Souza, C.R. Rodrigues, H.C. Castro, I.N. Junior, M.C.S. Lourenco, S.M.S.V. Wardella, V.F. Ferreira, Synthesis, 24

Journal Pre-proof

tuberculosis inhibitory activity, and SAR study of

N-substituted-phenyl-1,2,3-triazole

derivatives, Bioorg. Med. Chem. 14 (2006) 8644–8653. https://doi.org/ 10.1016/j.bmc.2006. 08.019. [67] S.G. Agalave, S.R. Maujan, V.S. Pore, Click Chemistry: 1,2,3-triazole as pharmacophore, Chem. Asian J.

4 (2011) 2696-2718. https://doi.org/ 10.1002/asia.

201100432. [68] Y. Li, Y. Y.Liu, N. Q. Chen, K. Z. Lü, X. H. Xiong, J. Li, One-pot regioselective synthesis of novel oximino ester-containing 1-aryl-4-chloro-3-oxypyrazoles as potential 97 (2014) 1269–1282. http://dx.doi.org/10.1002/hlca.

of

fungicides, Helv. Chim. Acta 201300407.

ro

[69] L. Qing, T. Wenqiang , Zh. Caili, G. Guodong, G. Zhanyong, Synthesis of water soluble chitosan derivatives with halogeno-1,2,3-triazole and their antifungal activity, Int. J. Biolog.

Jo

ur

na

lP

re

-p

Macromol. 91 (2016) 623–629. http://dx.doi.org/10.1016/j.ijbiomac.2016.06.006.

25

Journal Pre-proof

Table 1. Characteristics of polysaccharide derivatives Polymer’s code Substitution degree* Weight content of (mol/100 UGl)

pendant groups

CAC

d (nm)

(mg/dL)

Dex40-T-Cl

43

Dex40-T-Br

41.7

Dex40-T-H

41

ro

45.5

5.9

259

39. 5

7.4

271

-p

Dex40-T-F

41.9

39.8

6.2

263

43

4.1

246

35.1

9.5

295

36.4

8.2

284

re

47.9

lP

Dex40-T-OCH3

of

(wt%)

40.7

na

Dex40-T-CH3

Jo

ur

* Degree of substitution is expressed as the molar contents of pendant group per 100 glucopyranosidic units (UGl) d -mean diameter in Millipore water determined by DLS studies

26

Journal Pre-proof

Table 2. Antibacterial and antifungal activities of the tested compounds

Diameter of inhibition zones (mm)

11.10.05 13.30.57 14.10.05

0 0 100.05

Ciprofloxacin (5 µg/disc)

27.70.06

31.50.50

n.t*

n.t*

29.00.00

n.t*

n.t*

n.t*

30.00.00

28.00.00

n.t*

31.50.50

32.50.50

re

lP n.t*

ur

na

n.t*

Jo

Fluconazol (25 µg/disc) Voriconazol (1 µg/disc) n.t*=not tested

17.00.02 13.10.05 120.00

C. albicans ATCC 14053 14.00.00 n.t* n.t*

of

Dex-T-H Dex-T-CH3 Dex-T-OCH3

P. aeruginosa ATCC 27853 0 0 110.05

27

C. albicans ATCC 90028

ro

E. coli ATCC 25922

-p

Compounds

S. aureus ATCC 25923

Journal Pre-proof

Captions to Schemes and Figures

Scheme 1. Synthetic route for Dex40-T-A. Polymer chemical structure indicate the

of

preferential substitution of the polysaccharide at C2’ (A = H(a), F(b), Cl (c), Br (d), CH3 (e),

13

C NMR spectrum of Dex40-T-CH3 in DMSO-d6

-p

Fig. 1.

ro

OCH3 (f))

re

Fig. 2. Thermal decomposition curves of Dex-T-A

lP

Fig. 3. DSC thermograms for polysaccharide derivatives at second heating

na

Fig. 4. Difractograms for Dex40-T-A at room temperature

ur

Fig. 5. Variation of the intensity ratio I1/I3 with the polymer concentration for Dex40-T-H

Jo

Fig. 6. Size distribution measured by DLS for Dex40-T-H self-aggregates in Millipore water Fig. 7. TEM image of Dex40-T-H self-aggregates in Millipore water

Author statement Because the polymers synthesis was done within a project, data will be made available on request.

Highlights  

Dextrans with 1,4-disubstituted 1,2,3-triazoles as side-chains were obtained 13C-NMR, FTIR, thermal studies and WAXD were used for esters characterization

28

Journal Pre-proof

of ro -p re lP na ur

 

Polymeric self-associates, formed in aqueous media, were studied by DLS and TEM CAC for the dextran esters demonstrated lower values (4.1-9.5 mg/dL) Some of the polymers showed a moderate to good antimicrobial activity

Jo



29

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7