# Selective acylation of chitosan oligomers by several cyclic anhydrides as a 13C NMR quantification method

Paul Morandi, Steve Berthalon, Ghislain David, Aurelien Lebrun, Karine Parra, Claire Negrell

## ► To cite this version:

Paul Morandi, Steve Berthalon, Ghislain David, Aurelien Lebrun, Karine Parra, et al., Selective acylation of chitosan oligomers by several cyclic anhydrides as a 13C NMR quantification method. Carbohydrate Polymer Technologies and Applications, 2024, 7, pp.100498. 10.1016/j.carpta.2024.100498 hal-04990543

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Submitted on 22 May 2025

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# Selective acylation of chitosan oligomers by several cyclic anhydrides as a ¹³C NMR quantification method

Paul Morandi<sup>a</sup>, Steve Berthalon<sup>a</sup>, Ghislain David<sup>a</sup>, Aurelien Lebrun<sup>b</sup>, Karine Parra<sup>b</sup>, Claire Negrell<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Chitooligosaccharides (COS)

Glucosamine

Amine protection and deprotection

Acylation

Anhydrides

¹³C NMR quantification

## ABSTRACT

The chemistry of chitosan is a promising way to afford biobased and biodegradable complex materials using highly reactive compounds such as anhydrides. However, the potential applications are limited due to the absence of control over the acylation and the lack of precise characterization. After a model study on glucosamine, a selective acylation method of amines or alcohols of chitosan oligomers using anhydrides has been developed. The N-acylation of chitooligosaccharides has been achieved using several anhydrides and the O-acylation has been achieved in three steps: (1) Amine protection using *p*-anisaldehyde (2) O-acylation (with four anhydrides), and (3) Amine deprotection by removing the protective anisyl groups. Functionalized chitooligosaccharides have been characterized by a precise quantification method using ¹³C nuclear magnetic resonance spectroscopy. The resulting degrees of substitution show in most of the cases a stoichiometric reaction between anhydrides and amines of chitooligosaccharides, and around 70 % of efficiency for O-acylation, proving the promising potential of such modifications. To the best of our knowledge, we describe here the first example of selective acylation of chitooligosaccharides using anhydrides, and most of all the first example of ¹³C quantitative NMR spectroscopy performed on chitooligosaccharides and its derivatives. These innovative structures are the gateway to the creation of new biosourced and/or biodegradable surfactants.

## 1. Introduction

Over the years, environmental issues around the world have become more significant, and the production of polymer materials from petroleum derivatives is one of the biggest concerns in this context. The ecological transition being necessary, scientists began to think differently and started looking for any possible application using eco-friendly materials, despite the related constraints. Following this idea, bio-based polymers, such as cellulose, collagen, starch, or chitosan, have been studied in literature (Al-Hazmi et al., 2024; Nasrollahzadeh et al., 2020).

Chitosan is an eco-friendly polymer derived from chitin, the second most abundant natural polymer (after cellulose) that can be found in crustacean shells, insects and fungi (Aranaz et al., 2021; Bhattacharjee et al., 2020; Kou et al., 2022; Mohammad El-Aidie, 2018). Recently, it gained attention by being both bio-based and biodegradable, in addition to its capacity to be chemically modified in various ways (Shrestha et al., 2023). It is a linear polysaccharide composed of two repeating units

which are glucosamine (GlcN) and *N*-acetyl-glucosamine (GlcNAc), linked by a glycosidic bond. Its behavior (solubility and reactivity) greatly depends on its molecular weight and degree of acetylation "DA" (ratio of GlcNAc units on all units of the chain) (Aranaz et al., 2021; Bhattacharjee et al., 2020; Kou et al., 2022; Mohammad El-Aidie, 2018). Because of its antimicrobial (Bhattacharjee et al., 2020; Kou et al., 2022; Mohammad El-Aidie, 2018) and antioxidative properties (Aranaz et al., 2021; Mohammad El-Aidie, 2018), chitosan has been investigated for its possible applications in the food industry and agrochemistry (Aranaz et al., 2021; Meyer-Déru et al., 2022; Morin-Crini et al., 2019). Due to their biocompatibility, anti-inflammatory properties (Aranaz et al., 2021) and/or antitumor effects, chitosan and its derivatives have great potential in medicine such as surgery and tissue engineering (Kou et al., 2022), drug delivery (Aranaz et al., 2021) or oncology (Kou et al., 2022). It also has applications in various fields such as cosmetics (Morin-Crini et al., 2019), bitumen (Chapelle et al., 2021a) or water treatment (Holmes et al., 2023; Yang et al., 2016).

<footnote><sup>a</sup> ICGM, University Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote><sup>b</sup> LMP, University Montpellier, CNRS, Montpellier, France</footnote>

<footnote>* Corresponding author: Postal address: ICGM - UMR5253, Pôle Chimie Balard Recherche, 1919 route de Mende, 34293 Montpellier, France.
E-mail address: claire.negrell@enscm.fr (C. Negrell).</footnote>
The presence of both reactive OH and NH₂ sites on each single unit of chitosan enables various chemical modifications to afford new properties to chitosan and/or allow further chemical modifications for the synthesis of complex materials. Madera-Santana et al. (2018) recently reviewed the different pathways for efficiently grafting chitosan by using carboxylic acid, epoxy, alkene and many other groups. Among them, anhydrides are specifically interesting: first because they can react on both amine and alcohol quantitatively, and secondly because of the choice of anhydrides carrying non-polar chains that can lead to chitosan-based amphiphilic materials.

Few papers introduce some crude chitosan's modifications with cyclic anhydrides such as maleic anhydride (Don & Chen, 2005; Gopal Reddi et al., 2017; Zhang et al., 2007), succinic anhydride (Ranjbar-Mohammadi et al., 2010) or phthalic anhydride (Braz et al., 2020; Ifuku et al., 2011; Ranjbar-Mohammadi et al., 2010) to improve pervaporation, antibacterial or removal properties, or to bring carboxylic functions. The characterization of polysaccharides is still scarce, because of their complex sugar structure, presence of heteroatoms, high viscosity (depending on the molecular weight) and low solubility (Yao et al., 2021). Pure chitosan has already been well studied by Nuclear Magnetic Resonance (NMR) spectroscopy (Mourya et al., 2011; Vårum et al., 1991), but when modified with anhydrides its behavior leads to imprecise analyses. Acylation efficiency was only confirmed by infra-red (FTIR) analyses from which amide function is evidenced by the presence of a characteristic carbonyl peak (Braz et al., 2020; Don & Chen, 2005; Gopal Reddi et al., 2017; Ifuku et al., 2011; Ranjbar-Mohammadi et al., 2010; Zhang et al., 2007). Some ¹³C NMR analyses of modified chitosan were also performed (Alvarez Echazú et al., 2022; Braz et al., 2020; Don & Chen, 2005) to confirm anhydrides reaction, but most of the spectra lack resolution due to the high molecular weight of used chitosan. Ifuku et al. (2011) reported a quantitative ¹H NMR method of a chemoselective N-phthaloyl chitosan in aqueous media by comparing the isolated proton's peaks of the phthaloyl groups and the proton's peaks of glucosamine ring. This quantitative ¹H NMR method works for phthalic anhydride reaction thanks to the aromatic ring but lacks precision for other anhydrides due to overlapping peaks. In any case, neither FTIR and ¹³C NMR analyses are able to quantitatively assess the acylation efficiency, and quantitative ¹H NMR analyses are limited to specific anhydrides as aromatic-bearing anhydride (Ifuku et al., 2011). To the authors' knowledge, only two examples of acylation quantification using anhydrides were found in the literature: by elemental analysis (Sousa et al., 2022) and by comparing the product weight before and after acylation (Hasipoglu et al., 2005), but these methods remain very approximate. To date, there is no robust and universal method to characterize and quantify chitosan acylation using anhydrides, both on NH₂ and OH sites.

Recently, chitooligosaccharides (COS) and their derivatives have been studied (Chapelle et al., 2021b) and these have shown an improvement in their properties such as better antimicrobial, anti-tumor, anti-inflammatory and antioxidant activities (Chot-phruethipong et al., 2023; Naveed et al., 2019). Thanks to their shorter hydrophilic chain, their functionalization can lead to various structures such as nanomaterials (Nguyen et al., 2019), microgel (Fu et al., 2019), chelating agents (Zhang et al., 2011) or amphiphilic systems (Aranaz et al., 2010; Chapelle et al., 2021c). Unlike native chitosan and due to their low molecular weight, COS is completely soluble in water and in some polar organic solvents, which enables efficient chemical modifications. Furthermore, these COS are less viscous and more soluble in deuterated solvents, thus leading to an easier NMR spectroscopy characterization allowing a quantification of the functionalization. Thus, COS can be used as model compounds to understand the acylation of chitosan using anhydrides.

Here we propose a simple method for selective acylation of COS using various functional anhydrides, in addition to a quantification method. First, a study has been carried out on model molecules to conceive a strategy for selective acylation of glucosamine units. The

aims are to subsequently apply this strategy to COS, and then use a precise NMR spectroscopy method to quantify the acylation. As seen in the literature, we expect to modify only amine groups when pure COS are used (Braz et al., 2020; Don & Chen, 2005; Gopal Reddi et al., 2017; Ifuku et al., 2011; Ranjbar-Mohammadi et al., 2010; Zhang et al., 2007). For the same reason, we propose another way to perform selective O-acylation, involving a protection/deprotection method for keeping free amines, based on the primary study on model molecules. For all anhydrides tested, various acylation ratios have been set in order to assess COS reactivity, depending on the targeted site. All modified COS samples have been analyzed by ¹H NMR and 2D ¹H–¹³C HSQC NMR. Finally, ¹³C NMR with a helium cryoprobe was used to provide a precise quantification method for modified COS. This work is the first step towards the synthesis of new bio-based compounds having a specific ionic character, which can be tuned by controlling the O/N-acylation as well as the degree of substitution for each acylation. Indeed, according to the pKa of both carboxylic acid and amine groups, 4.8 and 6.5 respectively, the modified oligomers will show either a cationic, a zwitterionic (amphoteric), or an anionic character. The ionic strength of the oligomer will therefore depend on the degree of substitution for both O/N-acylation.

## 2. Materials and methods

Chitooligosaccharides called COS (M<sub>w</sub>: 1400 g.mol<sup>-1</sup> (DPn = 10) see SI, Fig. S11, Degree of acetylation "DA": 15 %) were obtained from VNF (Hô Chi Minh City, Vietnam). Glucosamine hydrochloride, *p*-anisaldehyde, acetic anhydride (AA), maleic anhydride (MA), succinic anhydride (SA), phthalic anhydride (PA), pyridine and anhydrous sodium acetate were purchased from Sigma Aldrich, and allyl succinic anhydride (ASA) from Gelest. Triethylamine, dichloromethane, ethanol and acetone were purchased from Carlo Erba, methanol from LabKem and diethyl ether from Honeywell. Hydrochloric acid was obtained from Fluka and sodium hydroxide from VWR. Ethyl acetate was obtained from Fisher. Dimethylsulfoxide (DMSO) was purchased from Fluka Chemicals. Finally, deuterated solvents for NMR were purchased from Eurisotop and Innovachem

### 2.1. Synthesis of 2-(4-Methoxybenzylidene)imino-2-deoxy-D-glucopyranose (1)

1 was synthetized via a NH₂ protection reaction of GlcAm·HCl with *p*-anisaldehyde according to a method described by Cunha et al. (1999). *p*-anisaldehyde (564 µL, 1 eq) was added to a stirred mixture of glucosamine hydrochloride (1.0 g, 1 eq) and 1 N aqueous sodium hydroxide solution (5 ml). Vigorous stirring was maintained for 2 h at room temperature (≈ 25 °C) and the product was collected by filtration, washed with cold water, diethyl ether / ethanol (1:1), and vacuum dried at 60 °C to produce 1 as a white solid (yield = 82 %).

¹H NMR (DMSO-d₆, 400 MHz) δ [ppm] (see SI, **Figure S1**): 8.11 (s, 1H, Hg), 7.68 (d, 2H, Hh, *J* = 8.7 Hz), 6.98 (d, 2H, Hi, *J* = 8.7 Hz)), 6.51 (OH), 4.95 (OH), 4.82 (OH), 4.69 (d, 1H, Ha, *J* = 7.7 Hz), 4.55 (OH), 3.79 (s, 3H, Hj), 3.71–3.49 (d, d, 2H, Hf), 3.42 (t, 1H, Hc, *J* = 7.0 Hz), 3.22 (t, 1H, He), 3.14 (t, 1H, Hd), 2.79 (t, 1H, Hb, *J* = 6.7 Hz).

### 2.2. Synthesis of 2-(4-Methoxybenzylidene)imino-2-deoxy-1,3,4,6-tetra-O-acetyl-D-glucopyranose (2)

2 was prepared by O-acylation of 1 according to a method described by Cunha et al. (1999). First, acetic anhydride (1.59 mL, 10 eq) was added to a stirred dispersion of 1 (0.5 g, 1 eq) in pyridine (2.7 mL, 2 eq) at 0 °C. The resulting mixture was stirred at this temperature until dissolution was complete. It was then kept at room temperature (≈ 25 °C) with stirring for 16 h. The solution was poured into cold water (7 mL) and left in the refrigerator (4 °C) for 1 h. After crystallization, the resulting solid was collected by filtration and washed with cold water,
then recrystallized in ethanol (7 mL) at 60 °C, filtered, washed with cold water again and vacuum dried to afforded **2** (yield = 47 %).

¹H NMR (CDCl₃, 400 MHz) δ [ppm] (see SI, **Figure S2**): 8.14 (s, 1H, Hg), 7.73 (d, 2H, Hh, *J* = 8.6 Hz), 6.93 (d, 2H, Hi, *J* = 8.6 Hz), 6.00 (d, 1H, Ha, *J* = 8.4 Hz), 5.47 (t, 1H, Hc, *J* = 9.8 Hz), 5.14 (t, 1H, Hd, *J* = 9.8 Hz), 4.40–4.11 (m, 2H, Hf, *J* = 2.12, 4.8 and 12.5 Hz), 3.99 (m, 1H, He, *J* = 4.5 and 10.2 Hz), 3.85 (s, 3H, Hj), 3.50 (t, 1H, Hb, *J* = 7.1 Hz), 2.10–1.89 (m, 12H, Hk/Hl/Hm/Hn).

## 2.3. Synthesis of 2-Amino-2-deoxy-1,3,4,6-tetra-O-acetyl-D-glucopyranosyl hydrochloride (3)

**3** was synthesized by a NH₂ deprotection reaction of **2** with HCl according to a method described by Cunha et al. (1999). GlcIm-A-n-(OAc)₄ (0.5 g, 1 eq) was dissolved in acetone (8 mL) heated at 60 °C under reflux, and 1 M hydrochloride acid solution (1.07 mL, 1 eq) was added dropwise. Stirring was maintained for 0.5 h and then the solution was cooled to room temperature (≈ 25 °C). Diethyl ether (10 mL) was added to it, and then it was kept in the refrigerator (4 °C) until crystallization. The resulting product was collected by filtration, washed with diethyl ether and vacuum dried at 60 °C to produce **3** (yield = 57 %).

¹H NMR (D₂O, 400 MHz) δ [ppm] (see SI, **Figure S4**): 5.98 (d, 1H, Ha, *J* = 8.8 Hz), 5.50 (t, 1H, Hc, *J* = 9.4 and 11.4 Hz), 5.16 (t, 1H, Hd, *J* = 9.4 and 10.8 Hz), 4.42–4.21 (m, 2H, Hf, *J* = 4.4 Hz), 4.22 (m, 1H, He, *J* = 2.5 and 10.8 Hz), 3.78 (t, 1H, Hb, *J* = 8.7 and 11.4 Hz), 2.24–2.12 (m, 12H, Hg/Hh/Hi/Hj).

## 2.4. Synthesis of 2-(1-Allyl-carboxyethyl)amido-2-deoxy-1,3,4,6-tetra-O-acetyl-D-glucopyranose (4)

**4** was synthesized by N-acylation of **3** using allyl succinic anhydride. First, **3** (1 g, 1 eq) was dissolved in ethyl acetate (10 mL) at 0 °C with stirring. Triethylamine (908 µL, 2.5 eq) was added into it and the reaction was maintained for 0.5 h at room temperature (≈ 25 °C). Then ASA (313 µL, 1 eq) was added and the mixture was maintained for 24 h more. The precipitated by-product (Et₃NHCl) was eliminated by filtration, and the filtrate was vacuum dried to obtain the product. It was then dissolved in ethyl acetate (20 mL) and this organic phase was washed with water (3 × 10 mL), HCl 1 M (3 × 10 mL) and water again (3 × 10 mL). Solvent was eliminated by vacuum dried and the resulting solid was washed with diethyl ether, vacuum dried at 60 °C and purified by flash chromatography with dichloromethane / methanol (93/7) to produce **4** (yield = 35 %).

¹H NMR (CDCl₃, 400 MHz) δ [ppm] (see SI, **Figure S6**): 6.58–6.41 (m, 1H, NH), 5.73 (m, 2H, Hn/Hn'/Ha), 5.35 (m, 1H, Hc), 5.06 (m, 3H, Ho/Ho'/Hp/Hp'/Hd), 4.35 (m, 1H, Hb), 4.25–4.13 (m, 2H, Hf), 3.93 (m, 1H, He), 2.98 (m, 1H, Hl/Hl'), 2.71–2.29 (m, 4H, Hk/Hk'/Hm/Hm'), 2.08–2.03 (m, 12H, Hg/Hh/Hi/Hj).

## 2.5. Synthesis of 2-(1-Allyl-dicarboximide)-2-deoxy-1,3,4,6-tetra-O-acetyl-D-glucopyranose (5)

**5** was prepared by a cyclisation reaction of ASA from **4**. This synthesis was inspired by a method described by Shetgiri and Nayak (2005). **4** (358 mg, 1 eq), acetic anhydride (69.5 µL, 1 eq) and anhydrous sodium acetate (61 mg, 1 eq) were dissolved in acetone (5 mL). The solution was stirred for 24 h at 100 °C under reflux. The precipitated by-product (AcONa) was eliminated by filtration and solvent was evaporated to obtain the desired product. This product was then purified by flash chromatography with dichloromethane / methanol (98/2) to afford **5** (yield = 55 %).

¹H NMR (CDCl₃, 400 MHz) δ [ppm] (see SI, **Figure S9**): 6.45 (d, 1H, Ha), 5.75 (m, 1H, Hc), 5.63 (m, 1H, Hn), 5.10 (m, 3H, Ho/Hp/Hd), 4.31–4.10 (m, 2H, Hf), 4.30 (m, 1H, Hb), 3.94 (m, 1H, He), 2.84 (m, 1H, Hl), 2.73–2.32 (m, 4H, Hk/Hm), 2.09–2.03 (m, 12H, Hg/Hh/Hi/Hj).

## 2.6. General procedure for the synthesis of N-acylated COS ("COS-N-Anhydride-eq")

All COS-N-Anhydride-eq were synthesized following the same method. A typical experiment for the synthesis of COS-N-SA-0.5 is reported hereafter, the amounts of reagents for the other COS-N-Anhydride-eq syntheses are summarized in SI (see SI). First, COS (3 g) was dissolved in DMSO (30 mL) at 50 °C with triethylamine (2.12 mL, 1eq/NH₂ of COS, nₙH₂ being calculated following the Eq. S1, see SI). Succinic anhydride (761 mg, 0.5 eq/NH₂ of COS) was added to the solution and maintained at 50 °C for 12 h with stirring. The resulting product was precipitated in methanol, collected by filtration, washed with the non-solvent and then vacuum dried at 60 °C to obtain COS-N-SA-0.5 (yield = 30%).

¹H NMR (D₂O, 400 MHz) δ [ppm] (see SI, **Figure S15**): 5.12 (Hb'), 4.51 (Hb/Hb''), 4.00–3.30 (Ha'/Ha''/Hc/Hd/He/Hf), 3.08 (Ha), 2.46 (Hh/Hi), 2.00 (Hg).

¹³C NMR (D₂O, 125 MHz) δ [ppm] (see SI, **Figure S17**): 178.12 (Cl), 175.37 (Ci), 174.52 (Cg), 97.45 (Cb), 90.50 (Cb'), 78.17–69.60 (Cc/Ce/Cf), 59.89 (Cd), 54.99 (Ca/Ca'), 30.84 (Cj/Ck), 22.00 (Ch)

## 2.7. Synthesis of COS-Anisyl ("COS-An")

COS-An was synthesized via a NH₂ protection reaction of COS with *p*-anisaldehyde. COS (5 g) was dissolved in 1 N aqueous sodium hydroxide solution (25 mL) and then *p*-anisaldehyde (6.99 g, 2 eq/NH₂ of COS, nₙH₂ being calculated following the Eq. S1, see SI) was added to the mixture. Vigorous stirring was maintained for 12 h at room temperature (≈ 25 °C). The product was precipitated in acetone, collected by filtration, washed with cold acetone, ethanol and diethyl ether, and vacuum-dried at 60 °C to afford COS-An (yield = 60 %).

¹H NMR (DMSO-d₆, 400 MHz) δ [ppm] (see SI, **Figure S46**): 8.14 (Hi), 7.69 (Hj), 6.97 (Hk), 4.70 (Ha/Ha'), 3.80 (Hl), 3.49–3.15 (Hb'/Hc/Hc'/Hd/Hd'/He/He'/Hf/Hf'), 2.88 (Hb), 1.81 (Hm).

## 2.8. General procedure for the synthesis of O-acylated COS-An ("COS-An-O-Anhydride-eq")

All COS-An-O-Anhydride-eq were synthesized following the same method. A typical experiment for the synthesis of COS-An-O-SA-0.5 is reported hereafter, the amounts of reagents for the other COS-An-O-Anhydride-eq syntheses are summarized in SI (see SI, **Table S2**). First, COS-An (1 g) was dissolved in DMSO (10 mL) at 50 °C with triethylamine (519 µL, 1 eq/repeating unit of COS-An, nₚGlcI/GlcNAc being calculated following the Eq. S2, see SI). Succinic anhydride (186 mg, 0.5 eq/repeating unit of COS-An) was added to the solution and maintained at 50 °C for 12 h with stirring. The resulting product was precipitated in acetone, collected by filtration, washed with acetone and then vacuum-dried at 60 °C to obtain COS-An-O-SA-0.5 (yield = 80 %).

¹H NMR (DMSO-d₆, 400 MHz) δ [ppm] (see SI, **Figure S50**): 8.14 (Hi), 7.68 (Hj), 6.98 (Hk), 4.70 (Ha/Ha'), 3.80 (Hl), 3.49–3.15 (Hb'/Hc/Hc'/Hd/Hd'/He/He'/Hf/Hf'), 2.88 (Ha), 2.30 (Hm/Hn), 1.78 (Hh)

## 2.9. General procedure for the synthesis of O-acylated COS ("COS-O-Anhydride-eq")

All COS-O-Anhydride-eq were synthesized by the removal of anisyl group in the corresponding COS-An-O-Anhydride-eq, following the same method. A typical experiment for the synthesis of COS-O-SA-0.5 is reported hereafter, the amounts of reagents for the other COS-O-Anhydride-eq syntheses are summarized in SI (see SI, **Table S3**). First, COS-An-O-SA-0.5 (1 g) was dissolved in H₂O (20 mL) at 50 °C, then 12 M aqueous HCl (225 µL, 1eq/Anisyl of COS-An-O-SA-0.5, nₙAnisyl being calculated following the Eq. S3, see SI) was added dropwise and the solution was stirring 2 h at 50 °C. The crude product was obtained by diluting the reaction media in MeOH, and all the solvents were removed
under vacuum. The final product was then obtained by solubilization in DMSO, precipitation in dichloromethane (DCM), collected by filtration, washed with DCM and finally dried under vacuum at 60 °C to afforded COS-O-SA-0.5 (yield = 30%).

¹H NMR (D₂O, 500 MHz) δ [ppm] (see SI, **Figure S77**): 5.5–5.2 (Hg/Hg'), 4.6–4.4 (Hb/Hb'/Hf'), 4.4–3.4 (Ha'/Hc/Hd/Hd'/He/Hf), 3.21 (Ha), 2.8–2.5 (Hi/Hj), 2.1 (Hh)

¹³C NMR (D₂O, 125 MHz) δ [ppm] (see SI, **Figure S78**): 178–174 (Ch/Cj/Cm), 101.20 (Cb'), 97.46 (Cb), 91–88 (Cg/Cg'), 80–67 (Cc/Ce/Cf/Cf'), 62.76 (Cd'), 60 (Cd), 55.79 (Ca), 54 (Ca'), 31–29 (Ck/Cl), 22.15 (Ci)

## 2.10. NMR characterization of model molecules (from 1 to 5), “COS-An” and “COS-An-O-Anhydride-eq”

¹H and 2D NMR spectra were recorded at 298 K on both Bruker Avance III 500 MHz NMR spectrometer using TCI Cryoprobe Prodigy® or Bruker Avance III HD 400 MHz spectrometer equipped with BBI probe. Chemical shift data are given in δ ppm calibrated with residual protic solvent (e.g. D₂O: 4.79 ppm –¹H). In addition, 2D homonuclear ¹H–¹H g-COSY (2 scans, 512 real (t1) × 2048 (t2) complex data points) and 2D heteronuclear spectra ¹³C–¹H g-edited HSQC (8–96 scans, 128–512 real (t1) × 2048 (t2) complex data points) were acquired to assign the compound (16 scans, 512 real (t1) × 2048 (t2) complex data points). Spectra were processed and visualized with Topspin 3.6.2 (Bruker Biospin) on a Linux station.

## 2.11. ¹H and 2D NMR characterization of “COS-N-Anhydride-eq” and “COS-O-Anhydride-eq”

¹H and 2D NMR spectra were recorded at 298 K on a Bruker AVANCE III 500 NMR spectrometer operating at 500.17 MHz equipped with a 5 mm ¹H/X BBO cryoprobe Helium. All the data were processed with Bruker Topspin 3.6.2 software. The pulse sequence used is a sequence with decoupling during acquisition, multiplicity editing during selection step and phase sensitive using echo/antiecho. For 2D ¹H/¹³C-spectra, the spectral widths are 5000 Hz and 20,833 Hz in ¹H and ¹³C dimensions, respectively. The experiments were acquired using 16 steady state scans, 32 transients and data matrix size of 2048 (0.21 s acquisition time).

## 2.12. Quantitative ¹³C NMR characterization of “COS-N-Anhydride-eq” and “COS-O-Anhydride-eq”

¹³C NMR spectra were recorded at 298 K on a Bruker AVANCE III 500 NMR spectrometer operating at 500.17 MHz equipped with a 5 mm ¹H/X BBO cryoprobe Helium. All the data were processed with Bruker Topspin 3.6.2 software. The pulse sequence used is a sequence with inverse gated decoupling. Chromium(III) acetyl acetonate Cr(acac)₃ was used as a relaxation agent leading to a relaxation delay of 10 s and 4096 scans.

# 3. Results and discussion

## 3.1. Model synthesis

The chemical modification of chitosan is always a challenge. Its polysaccharide nature leads to obvious synthesis constraints such as low solvent solubility, low reactivity or multiple reaction sites (in the case of reaction with anhydride). Assessing the modification efficiency can also be hard to achieve, once more due to its polysaccharide nature. For those reasons, a model synthesis has been made using glucosamine hydrochloride (GlcAm·HCl), which represents a major part of chitosan repeating units (Scheme 1).

The purpose of this model synthesis was to develop a strategy for selective acylation for subsequently applying it to COS. The obtained products will help us to assess the characterization of anhydride-modified COS. This synthesis is based on the work of Cunha et al. (1999), and is suitable to our goal for multiple reasons, such as the first protection step made in aqueous conditions, or the second step involving an anhydride (which can be further adapted with multiple anhydrides onto COS).

The amine protection/deprotection process involves the use of *p*-anisaldehyde, which is a real contribution when applying this process to COS. Indeed, the chemical shift of the aromatic ring of *p*-anisaldehyde in a ¹H NMR spectrum can be easily isolated and used to prove the amine full protection (see SI, **Figure S1**). By comparing the integrations of the signal corresponding to the imine proton (HC–N=CH) and the signal corresponding to the glucosamine ring proton in the α-position of the nitrogen (HC–N=CH), the degree of protection can be calculated. In this case, both integrations have the same value, meaning that no unprotected amines are left. In addition, 2D ¹H–¹³C HSQC NMR analyses have been carried out to observe the shift of the glucosamine ring proton

<figure><img src="image_4.png" /><figcaption>**Scheme 1.** Synthetic pathway for selective acylation model reaction onto glucosamine in four steps: A. Amine protection using *p*-anisaldehyde; B. O-acylation using acetic anhydride; C. Imine group removal and amine regeneration using hydrochloric acid; D. N-acylation using allyl succinic anhydride.</figcaption></figure>
and carbon in the α-position of the nitrogen (Fig. 1). Although the proton does not have a significant shift (from 3.1 to 2.8 ppm), the carbon shifted from 57 ppm to 78 ppm, which will be a good marker of the protection when applying this process on COS.

As seen in Fig. 1, glucosamine hydrochloride has an α and a β anomers, and p-anisaldehyde will selectively react on the β-anomer, due to the configuration which makes the NH₂ group more available than in the α-anomer because of the steric hindrance. In fact, the α-anomer of 1 is also present in the purified product, but in a much lower proportion compared to the β-anomer of 1, explaining why it cannot be seen properly in the ¹H–¹³C HSQC spectrum (Fig. 1). In our case, this is relevant because the glucosamine units in COS are actually in the β configuration, which allows us to not consider the α-anomer of glucosamine for the rest of the model synthesis.

The protection of hydroxyl groups using acetic anhydride is the model synthesis second step. Once more, this step is perfectly adapted to our goal: O-acylation of COS is possible without affecting any amine (because of the imine protection), in addition to protecting the hydroxyl group for further N-acylation in the case of the model synthesis. Indeed, hydroxyl protection is not necessary for N-acylation on COS due to the higher reactivity of amine groups (as seen in the literature (Braz et al., 2020; Don & Chen, 2005; Gopal Reddi et al., 2017; Ifuku et al., 2011; Ranjbar-Mohammadi et al., 2010; Zhang et al., 2007)), but it is probably required in the case of the model synthesis. Glucosamine hydrochloride bears 4 different hydroxyl groups (whereas repeating units of chitosan have only 2 hydroxyl functions) and doesn't have as much steric hindrance as on the chitosan polymer chain. Considering this, the full protection of the hydroxyl groups is mandatory to be sure that the acylation will be fully targeted on the nitrogen without any unwanted side reactions, in order to properly characterize the final product (2).

As seen in the ¹H NMR spectrum of the obtained 2 (see SI, **Figure S2**), anhydride has efficiently reacted on all hydroxyl groups of 1, without affecting the N-protective group. The ¹H–¹³C HSQC spectrum also confirms that the anisyl group is not affected by the O-acylation, as the carbon chemical shift of the imine is still around 70 ppm (see SI, **Figure S3**).

The third step in the model synthesis involves the removal of the anisyl group leading to amine regeneration. This protection/deprotection process involving *p*-anisaldehyde is particularly interesting for this work, owing to the deprotection step. Indeed, a typical amine protection using (Boc)₂O (Di-tert-butyl dicarbonate) could have been used,

but the deprotection process involves the use of TFA (Trifluoroacetic acid) in excess, which is a strong acid. In these conditions, the O-acylation may be affected by the TFA during the deprotection process. In our conditions, the anisyl group removal using a stoichiometric amount of hydrochloric acid allows us to completely regenerate the amine without affecting the O-acylation (see SI, **Figure S4**). As the chemical shift of the glucosamine ring proton in α-position of the nitrogen is barely the same before and after anisyl removal (respectively *HC*—*N*==*CH* and *HC*—*NH₂*·*HCl*, both around 3.5 ppm), a ¹H–¹³C HSQC NMR analysis has been made to confirm the carbon shift (*HC*—*N*==*CH* having a signal around 70 ppm and *HC*—*NH₂*·*HCl* around 55 ppm), proving the full regeneration of amine (see SI, **Figure S5**).

Finally, the last step of the model reaction is focused on the N-acylation. As the model synthesis aimed to help the characterization for further COS modifications, allyl succinic anhydride has been chosen instead of acetic anhydride, to avoid any NMR spectrum overlapping. Allyl succinic anhydride is not symmetrical, this reaction results in an isomer mixture of 4, depending on the anhydride cycle opening (see SI, **Figure S6**). To confirm this, a high-temperature dehydration reaction has been carried out in order to obtain a succinimide cycle, thus avoiding the isomer mixture. ¹H NMR spectrum of the product 5 confirms this assumption (see SI, **Figure S9**).

Yet, the NMR analyses of compound 4 confirm the full reaction of allyl succinic anhydride onto amine (Fig. 2, and see SI, **Figure S6**, **Figure S7** and **Figure S8**). This last step allows us to use the ¹³C NMR spectrum of 4 (Fig. 2) as a reference for the ¹³C NMR spectrum of acylated COS assignment, hence allowing acylation quantification.

When applying COS acylation using anhydrides, the signal of the glucosamine ring carbon in the α-position of the primary alcohol can be used as a reference peak. Indeed, even though the proton signal will shift in ¹H NMR before and after the anhydride reaction, the carbon won't shift a lot and will stay around 60 ppm (see respectively Fig. 1 and SI **Figure S5**). By calibrating the integration on this last carbon peak, the acylation efficiency can be easily determined using the carbon signals of the reacted anhydride. Taking the example of the allyl succinic anhydride as used in the model reaction, the carbons of the vinyl group are well isolated and can be an efficient marker (Fig. 2).

Each anhydride used in this work has its own isolated peaks on the ¹³C NMR spectrum that can be used to assess the acylation. It can be noticed that all anhydrides have carbonyl groups that can potentially be used, but there is a risk of overlapping with the carbonyl of N-acetyl-

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Overlay of ¹H–¹³C HSQC NMR spectrum (400 MHz) and assignment of GlcAm·HCl in D₂O (CH/CH₃ in red and CH₂ in blue) and the molecule 1 in DMSO-d₆ (CH/CH₃ in purple and CH₂ in green).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 2. ¹³C NMR spectrum (100 MHz, CDCl₃) and carbon assignment of 4.</figcaption></figure>

glucosamine (GlcNAc) units present in chitosan.

## 3.2. Preparation and characterization of N-acylated-COS

According to the literature (Braz et al., 2020; Don & Chen, 2005; Gopal Reddi et al., 2017; Ifuku et al., 2011; Ranjbar-Mohammadi et al., 2010; Zhang et al., 2007), the reaction of anhydrides onto unmodified COS will directly lead to amine groups acylation. Four different anhydrides have been used with pure COS, with anhydride/NH₂ molar ratio of 0.2, 0.5 and 1, allowing us to evaluate the reactivity of the N-acylation (see step A from Scheme 2). As the anhydride molar ratio is dependent on the amine groups of COS, the degree of acetylation (DA) has been determined by quantitative ¹³C NMR spectroscopy, using the same method that will be used to assess acylation efficiency. The resulting spectrum (see SI, Figure S10) indicates a DA close to 15 %.

First and foremost, we used succinic anhydride “SA” because its simple structure makes the NMR spectra interpretation easier. Indeed, the two CH₂ groups of SA in the α-position of the carbonyl can be spotted in both ¹H NMR and ¹³C NMR. Therefore, before using ¹³C NMR to precisely quantify the acylation, ¹H NMR spectra can be used to check if

the reaction took place (see SI, Figure S12). The signal of the CH₂ in the α-position of the carbonyl at 2.5 ppm proves the opening of the succinic anhydride cycle (whereas in pure succinic anhydride, these signals are around 2.9 ppm). In addition, the peak integration of the CH in the α-position of the amines at 3.1 ppm clearly decreases from 1 to 0.8 (calibrate using the acetyl peak at 2 ppm).

¹H-¹³C HSQC NMR analyses allowed assessing that the acylation was exclusively performed onto amine groups. By comparing the HSQC spectra of COS-N-SA-0.2, COS-N-SA-0.5 and COS-N-SA-1 (see SI, respectively Figure S13, Figure S16 and Figure S19), the increasing spots of CH in α-position of amide groups are clearly seen, and there are no signs of shifting concerning the CH₂ in α-position of alcohols. Nevertheless, a slight shift of CH₂ in the α-position of alcohols can be spotted in the carbon spectrum of COS-N-SA-1 (see SI, Figure S20), proving that the anhydride will also react onto alcohols after all amines have reacted. To confirm this hypothesis, acylation using succinic anhydride has been made with 1.5 equivalent compared to amine groups. The HSQC spectrum of the obtained product confirmed our expectation (see SI, Figure S21), as the proton signals of the CH₂ in α-position of primary alcohol are clearly shifting from 3.4 to 3.8 to 4-4.5 ppm, while

<figure><img src="image_7.png" /><figcaption>**Scheme 2.** Synthetic pathway for N-acylation (1 step-red way) or O-acylation (3 steps-green/blue way) of COS: A. N-Acylation of COS; B. Amine protection using p-anisaldehyde onto COS; C. O-acylation of COS-An; D. Regeneration of amines in COS-An-O-Anhydride.</figcaption></figure>
all the amines are converted to amides.

However, due to the width of the peaks and their overlapping, quantifying the acylation using the ¹H NMR spectrum is very approximate and lacks precision. According to the HSQC spectra of succinic anhydride modified COS (see SI, respectively Figure S13, Figure S16 and Figure S19), in correlation with the observations of the model synthesis, quantitative ¹³C NMR spectroscopy is a way more precise and can assess acylation efficiency.

Indeed, the carbon spectra are simpler than the corresponding proton spectra since they suffer from much fewer signal overlaps and have a better resolution. The aim was to obtain a spectrum displaying all the carbon resonances of the compound under investigation with a sufficient signal to noise (using a cryoprobe) and in a reasonable amount of time (adding a small amount of chromium(III) acetylacetonate).

Specific signals have then been selected purposely to calibrate all the integrations, which will directly lead to a proper acylation quantification (Fig. 3).

The carbon in the α-position of primary alcohol (60 ppm) is present on both glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) units, meaning that they can be all integrated and used as a reference for the COS backbone (with a value of 1). Then, the integration of the two CH₂ for the anhydride (spotted at 31–34 ppm) leads to a precise quantification of the acylation calculated thanks to the following equation (Eq. (1)):

$$NH_2 DS_{COS-N-SA}(\%) = \left( \frac{\int (C_j/C_k)/2}{\int C_d * [1 - DA]} \right) * 100 \quad (1)$$

Eq. (1) is the ratio between the number of reacted anhydride group and the backbone of the COS, more specifically the glucosamine units which represent the major part of the COS backbone. The value of “∫ (Cj/Ck)” is divided by 2 because the integration considers the two carbons of the succinic anhydride. As said previously, the carbons in the α-position of primary alcohol represent the whole COS chain, therefore the value of “∫ C₉” is used as the reference and is set to 1. Finally, “[1-DA]” is added to consider only glucosamine units of the backbone, because N-acetyl-glucosamine units are not affected by the anhydride. In our case, the degree of acetylation “DA” is equal to 15 %. For all anhydrides, the equation for the degree of substitution is nearly the same, just adapted to the carbon used for the quantification. Only COS modified with 1 equivalent of phthalic anhydride (COS-N-PA-1) could not

have been characterized, due to lack of solubility in the deuterated solvents. All the ¹H, HSQC and quantitative ¹³C NMR spectra of all N-acylated COS are given in supporting information (see SI, from Figure S12 to Figure S45). All the resulting degrees of substitution are gathered in Table 1.

The experimental degrees of substitution concerning the N-acylated COS show stoichiometric reactions for all the anhydrides using triethylamine as a catalyst. For maleic anhydride “MA” reactions, pyridine was used as a catalyst. Triethylamine was indeed not efficient, probably because maleic anhydride and triethylamine can form a complex in basic conditions, as already reported in the literature (Mayahi & El-Bermani, 1973). Nevertheless, the lower basicity of pyridine may explain the discrepancy between experimental and theoretical DS values. For the other anhydrides, the experimental DS is very close to the theoretical one, even slightly higher in some cases (52 % of amines in “COS-N-ASA-0.5” are acylated instead of the expected maximum value of 50 %). The main reason explaining this observation is related to the DA determination (determined using quantitative ¹³C NMR, see Figure S10). The N-acylation affecting only the glucosamine units of COS, the amount of anhydride used is directly related to the ratio of glucosamine units compared to the whole chain, i.e. “(1-DA)” (known as the degree of deacetylation “DD”). There is in fact a margin of error (around 5 %) from NMR explaining the slightly higher anhydride equivalent compared to amines. For the same reason and as explained before, a low reaction onto alcohols occurs when all the amines have reacted with anhydrides (in the case of “COS-N-SA-1” and “COS-N-ASA-1”, see SI respectively Figure S20 and Figure S45). In these specific cases, the degree of substitution for alcohols is calculated thanks to the following equation (Eq. (2)):

$$OH * DS_{COS-N-SA-1}(\%) = \left( \frac{\int (C_j/C_k)/2 - [1 - DA]}{\int C_d/C_d} \right) * 100 \quad (2)$$

Finally, it should be noticed that unlike succinic and allyl succinic anhydride (“SA” and “ASA”), the use of phthalic anhydride (“PA”) is slightly less efficient for the same reaction conditions. Indeed, the lower degree of substitution and the traces of unreacted phthalic anhydride in the NMR spectra (see SI, Figure S33 and Figure S36) confirm that phthalic anhydride did not totally react. The presence of the aromatic ring induces steric hindrance that may explain this lower reactivity.

<figure><img src="image_8.png" /><figcaption>Fig. 3. ¹³C NMR spectrum (D₂O, 125 MHz) of COS-N-SA-0.5 with the preferred signals for assessing the acylation.</figcaption></figure>
$^1$H-$^{13}$C HSQC spectrum does not show any alteration of the protective group, as the anisyl group is still present around 7–8 ppm and the proton at 3 ppm is attached to the carbon at 75 ppm (see SI, Figure S51), confirming the selective acylation of alcohols of COS-An. All the $^1$H and $^1$H-$^{13}$C HSQC NMR spectra of all O-acylated COS-An are given in supporting information (see SI, from Figure S48 to Figure S71)

The final O-acylated COS has then been obtained by removing the protecting group of the corresponding O-acylated COS-An, regenerating the amine groups of COS. This final step is adapted from the model synthesis (see step D in Scheme 2). Chlorohydric acid is used to remove the anisyl group, therefore the experimental conditions have been studied and carefully chosen to avoid any hydrolysis of the reacted anhydride. Indeed, the first test of deprotection has been done using a slight excess of HCl compared to anisyl group (molar ratio HCl/Anisyl was around 1.2), in addition to a high reagent concentration (1 g of O-acylated COS-An in 10 mL of H$_2$O). Although the deprotection worked, the analysis of the resulting product clearly shows an alteration of the previously used anhydride, proved by the presence of molecular succinic acid at 2.4 ppm (see SI, Figure S72 and Figure S73). By adding a stoichiometric amount of HCl (molar ratio HCl/Anisyl at 1), and performing the deprotection in diluted conditions (1 g of O-acylated COS-An in 20–30 mL of H$_2$O), the anisyl group was totally removed without affecting the previous O-acylation (see SI, Figure S77 and Figure S78). The final product was soluble in D$_2$O, allowing us to assess the O-acylation using quantitative $^{13}$C NMR spectroscopy (Fig. 4).

The O-acylation has been determined using the same methodology as the N-acylation: the carbon in α-position of the primary alcohol and the corresponding ester (CH$_2$-OH and CH$_2$-O-R at 60–62 ppm) were used as a reference to represent the COS backbone (with a value of 1), and the value of the used anhydride is measured from Fig. 4, the two CH$_2$ of the succinic anhydride were used. The degree of substitution is finally calculated thanks to the following equation (Eq. (3)):

$$OH \ DS_{COS-O-SA}(\%) = \left( \frac{\int Ck/Cl}{\int (Cd/Cd)} \right) * 100 \quad (3)$$

Eq. (3) is similar to Eq. (1) (for the N-acylation), as it is a ratio between the number of acylated units and the COS backbone. It should be noticed that unlike Eq. (1), the degree of acetylation “DA” is not considered here because the O-acylation can occur on all repeating units of COS (GlcN and GlcNAc). For all anhydrides, the equation for the degree of substitution is nearly the same, just adapted to the carbon used for the quantification. As for N-acylation, O-acylated COS with 1 equivalent of phthalic anhydride (COS-O-PA-1) could not have been

characterized, due to lack of solubility in the deuterated solvents. All the $^1$H, HSQC and quantitative $^{13}$C NMR spectra of all O-acylated COS are given in supporting information (see SI, from Figure S74 to Figure S106). All the resulting degrees of substitution are gathered in Table 1.

DS values are promising, as the average efficiency of the acylation is around 70 % (except for maleic anhydride whose reaction was catalyzed with pyridine). Compared to stoichiometric N-acylation, the efficiency difference can be explained by the fact that hydroxyl groups are less nucleophile than amines. Furthermore, the anisyl group in COS-An undergoes steric hindrance compared to pure COS. Surprisingly, the O-acylation of COS occurred on both primary and secondary alcohols, while primary alcohols are more reactive. This observation has been made through the $^1$H-$^{13}$C HSQC NMR spectrum of COS-O-SA-0.5 (see SI, Figure S78), where CH of glucosamine ring in the α-position of esterified secondary alcohol has been spotted at 4.4 ppm for $^1$H and 73 ppm for $^{13}$C. It is also confirmed by the quantitative $^{13}$C NMR spectrum of the resulting product (Fig. 4), where the signal of CH$_2$ in α-position of acylated primary alcohol (62 ppm) is clearly not as intense as the signal of the reacted anhydride (30 ppm). Considering that the O-acylation without NH$_2$ protection did not affect secondary alcohol (see SI, Figure S21), it is obvious that the anisyl group used for NH$_2$ protection played a key role in the O-acylation. Our hypothesis is the following: the presence of the aromatic ring additionally to the imine bond that constrains the movement of the anisyl group is limiting the access of the primary alcohol, considering that the primary hydroxyl and the amine (and so the anisyl group) are both in axial positions of the cycle (whereas secondary alcohol shows equatorial position to the cycle). We believe that this interesting observation could lead to further work on the chitosan protective group and functionalization of secondary alcohols.

## 4. Conclusion

A simple and efficient method for selective acylation of chitooligosaccharides has been developed, and the acylation has been assessed for every compound using an innovative quantification method based on $^{13}$C NMR spectroscopy. For such purpose, a preliminary model synthesis has been carried out, using glucosamine hydrochloride to represent chitosan repeating units. This model synthesis in four steps aimed at developing a strategy for the selective acylation and the $^{13}$C NMR characterization.

Then, acylated COS have been synthesized using various anhydrides. Following the literature, the N-acylation has been carried out directly

<figure><img src="image_9.png" /><figcaption>Fig. 4. $^{13}$C NMR spectrum (D$_2$O, 125 MHz) of COS-O-SA-0.5 with the preferred signals for assessing the acylation.</figcaption></figure>
with pure COS and the quantitative ¹³C NMR analyses showed for most of the cases an experimental degree of substitution very close to the theoretical one. The resulting N-acylated COS showed no trace of O-acylation, except when no free amine groups remained.

The O-acylation of COS has been carried out in three steps, using an amine protection/deprotection method. Although anhydrides revealed to be less reactive with alcohols than with amines, the average efficiency of the O-acylation is around 70 %, which can be expected with this type of reaction. Surprisingly, the acylation occurred not only on primary alcohols but also on secondary alcohols of COS, as revealed in the HSQC spectra. The presence of anisyl group (amine protection) obviously played a key role during this step.

To conclude, the obtained results demonstrate the high reactivity of anhydrides for both amine and alcohol acylation, thus proving the potential of this chemistry to synthesize bio-based amphiphilic compounds for various applications (Aranaz et al., 2010; Chapelle et al., 2021c). Moreover, we described here the first example of quantitative ¹³C NMR spectroscopy performed on COS. It would then be interesting to transpose and adapt these quantification methods to higher molecular weight chitosans, which could represent a step forward in the characterization of modified chitosans and derivatives.

### CRediT authorship contribution statement

**Paul Morandi:** Writing – original draft, Visualization, Validation, Methodology, Investigation, Conceptualization. **Steve Berthalon:** Writing – original draft, Visualization, Validation, Methodology, Investigation. **Ghislain David:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization. **Aurelien Lebrun:** Writing – original draft, Visualization, Validation, Methodology, Investigation. **Karine Parra:** Writing – original draft, Visualization, Validation, Investigation. **Claire Negrell:** Writing – review & editing, Validation, Supervision, Project administration.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### Associated content

Additional experimental details, including the amounts of reagents for the syntheses of N-acylated COS, O-acylated COS-An, O-acylated COS and the corresponding equations used to determine these amounts; NMR spectra (¹H, ¹³C, HSQC and/or COSY NMR spectra) of all compounds described in this paper.

### Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

### Acknoledgements

The authors gratefully acknowledge Vietnam Food (VNF) for providing the chitooligosaccharides used in this paper.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.carpta.2024.100498.

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