# Dual functionalization of carboxymethyl cellulose and alginate via Passerini three-component reaction to graft two hydrophobic moieties: Toward modular thin films

Clémence Vuillet, C. Le Mouël, Pierre Alcouffe, E. Fleury, Aurelia Charlot, David Albertini

► To cite this version:

Clémence Vuillet, C. Le Mouël, Pierre Alcouffe, E. Fleury, Aurelia Charlot, et al. Dual functionalization of carboxymethyl cellulose and alginate via Passerini three-component reaction to graft two hydrophobic moieties: Toward modular thin films. Carbohydrate Polymers, 2025, 351, pp.123066. 10.1016/j.carbpol.2024.123066. hal-04944864

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<figure><img src="image_1.png" /></figure>

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1 **Title**

2 Dual functionalization of carboxymethyl cellulose and alginate *via* Passerini three-component reaction to

3 graft two hydrophobic moieties: towards modular thin films

4 **Authors**

5 C. Vuillet,ª C. Le Mouël,ª D. Albertini,ᵇ P. Alcouffe,ª E. Fleury, *ª A. Charlot¹*ª

6 **Affiliation**

7 ª Université Claude Bernard Lyon 1, INSA Lyon, Université Jean Monnet, CNRS UMR 5223, Ingénierie des

8 Matériaux Polymères, F-69621 Cédex, France

9 b CNRS, INSA Lyon, École Centrale de Lyon, Université Claude Bernard Lyon 1, CPE Lyon, INL, UMR5270, 69621 Villeurbanne, France
10

11 **Abstract**

12 Passerini reaction was advantageously exploited to hydrophobize carboxymethyl cellulose (CMC) and
13 alginates (ALG) by employing various hydrophobic aldehydes and isocyanides. The Passerini reaction,
14 carried out in ecofriendly conditions, allowed to design never described twofold hydrophobized
15 polysaccharide derivatives *via* the covalent grafting of two hydrophobic moieties. The modified CMC and
16 ALG products were in-depth characterized to guaranty the success of the modification and to calculate the
17 degrees of substitution (DS). The impact of experimental parameters and especially the structure of the
18 aliphatic reactants were thoroughly discussed. It appears that high conversions in carboxylic acid up to 70%
19 can be reached. Finally, the Passerini CMC and ALG products were processed as thin films exhibiting
20 modular wettability properties varying from a moderate to a significant hydrophobicity adjustable by the
21 structure of the grafts and the DS values. The film formation of selected CMC and ALG samples was
22 examined by QCM-D experiments completed by AFM analysis under humid environment. It appears that
23 the functionalization *i*) increases the adsorbed mass by inducing a more packed deposition and *ii*) closely
24 governs the energy dissipation of the films. This overall approach paves the way toward new bio-based
25 multifunctional films with potential utilizations in coating fields.
26

26

27 **Keywords**

28 Passerini, Carboxymethyl Cellulose, Alginate, film, QCM-D, AFM in liquid.

29
## Twofold functionalization by Passerini reaction to graft two hydrophobic moieties

<figure><img src="image_2.png" /><figcaption>Alginate
derivatives</figcaption></figure>

<figure><img src="image_3.png" /></figure>

In Water
At RT

<figure><img src="image_4.png" /></figure>

## Hydrophobic thin films

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

31

## 32 Main Body

### 33 1. INTRODUCTION:

For various applications, renewable polymers are promising alternatives to petrobased polymers in the quest to reduce reliance on fossil fuel. Profusely found in plants, animals, or bacteria, polysaccharides are a very appealing class of natural polymers. Abundant, non-toxic, biodegradable and in some cases biocompatible, they are used in food, cosmetic and pharmaceutical fields (Benalaya et al. 2024). Polysaccharides carry reactive functional groups that can be advantageously chemically modified to afford new functional properties and to expand their scopes of utilizations. Among polysaccharides and their derivatives, carboxymethyl cellulose (CMC) and alginates (ALG) exhibit very attractive features. Both bear carboxylic acid groups, imparting them with high water-solubility and high reactivity, prone to be involved in various chemical functionalization routes. CMC is an industrially produced cellulose ether derivative (300 kT produced in 2001) (Heinze and Liebert 2001) and can be found with various degrees of carboxymethylation. Also, alginates are naturally carboxylated polysaccharides mainly extracted from the cell walls of brown macro-algae or from bacteria (Pettignano, Aguilera, et al. 2019) (23 kT extracted annually from 85 kT tons of algae) (Bertagnolli et al. 2014). Their linear anionic chains consist of β-(1→4)-linked D-mannuronic acid (M) and α-(1→4) linked L-guluronic acid (G) arranged in homogeneous (G-blocks, M-blocks) and heterogeneous sequences (MG-blocks) of various length. With the goal to enlarge their applicative potential, various reactions have been employed to modify CMC and ALG backbone. For instance, hydroxyl functions were modified by acetylation to reach gelling properties (Chamberlain, Cunningham, and Speakman 1946; Skjåk-Bræk, Paoletti, and Gianferrara 1989; Skjåk-Bræk, Zanetti, and Paoletti 1989; Wassermann 1946), by phosphorylation to develop injectable hydrogels (Coleman et al. 2011; Kim et al. 2015), or by an oxidation step and reductive amination to conceive surfactants (Andresen, Painter, and Smidsrød 1977; Carré et al. 1991; Kang, Jeon, et al. 2002; Kang, Shin, and Yang 2002). The -COOH groups have been conventionally subjected to esterification (Broderick et al. 2006; Leonard et al. 2004; Pawar and Edgar 2011; Pelletier et al.
56 2001; Rastello De Boisseson et al. 2004) or amidation (Abu-Rabeah et al. 2005; Batelaan and Horst 2000;
57 Charpentier et al. 1997; Čopíková et al. 2015; Galant et al. 2006; Gomez et al. 2006; Polyak, Geresh, and
58 Marks 2004; Tizzotti et al. 2010; Vallée et al. 2009; Yang et al. 2002; Zabivalova et al. 2003; Zhu et al.
59 2002). A recent review describes the alginate modification by click chemistries for utilizations in biomedical
60 field (Deng et al. 2021). Our team reviewed knowledge concerning modifications of CMC for the
61 development of advanced biomedical systems (Pettignano, Charlot, and Fleury 2019). In particular, the
62 grafting of hydrophobic moieties extends the final properties of the derivatives. In this way, enhanced
63 solubility in organic solvents can be attained (Pawar and Edgar 2013) and viscosifying or gelling effect can
64 be induced by hydrophobic interactions in water (Sinquin, Houzelle, et al. 1996; Sinquin, Hubert, et al. 1996).
65 Amphiphilic alginate-based products are prone to self-association yielding micelle-like aggregates (Chen et
66 al. 2021; Yang, Zhou, and He 2013). For example, the coupling of linear alkyl groups onto ALG chains
67 allowed to prepare colloidal objects in water (Kang, Shin, et al. 2002). Hydrophobized ALG derivatives have
68 been employed as carriers in drug delivery systems. By using esterification and amidation reactions,
69 Dellacherie et al. designed alginate beads bearing alkyl chains able to encapsulate therapeutic proteins
70 (Leonard et al. 2004; Vallée et al. 2009). Also, the grafting of aliphatic chains onto alginate, described by
71 Nyström et al., was advantageously exploited to interact with β-cyclodextrin molecules (Galant et al. 2006).
72 Hydrophobic ester derivatives of CMC found applications as clean-up sorbent or to extract target analysts
73 (Thabet et al. 2023).

74 However, most of these chemical reactions are achieved in organic solvents, generally require a large
75 excess of reagents, the use of coupling agents, such as carbodiimides, which leads to the formation of
76 difficulty eliminable by-products thus necessitating intensive purification steps. Moreover, the introduction
77 of various functionalities requires successive chemical modification steps. Consequently, the challenge is
78 to develop non-degrading functionalization pathways conjugating efficiency and sustainability, while
79 providing tailor-made (multi)functionalities. To tackle this challenge, multicomponent reactions (MCRs)
80 are attractive approaches, in which multiple precursors concomitantly react in “one-pot” manner, to form
81 original structures composed of most of the atoms of the starting precursors. In literature, work was
82 conducted on the use of Ugi four-component reaction, which involves an amine, a carboxylic acid, an
83 aldehyde and an isocyanide. It offers a versatile and effective chemical pathway to functionalize
84 polysaccharides and polysaccharide derivatives such as chitosan, hyaluronic acid, CMC, or alginate (Bu et
85 al. 2004; Gabriel and Heinze 2018; Nooy 1999). In particular, Passerini three-component reaction (P-3CR)
86 responds to many criteria of green chemistry, linked not only to its atom economy but also to its possible
87 implementation in aqueous conditions, without the use of any coupling reagent. P-3CR, involving a
88 carboxylic acid, a carbonyl and an isocyanide to form an α-acyloxycarboxamide ligation, has been
89 successfully employed for the polysaccharide derivatization. In 1999, Crescenzi et al. were the first to
90 apply P-3CR to polysaccharides (CMC, sodium hyaluronate...) to generate cross-linked hydrogels with
91 tunable properties (Nooy 1999). Twenty years later, Meier et al. gave a new lease of life to the field by
92 modifying - succinylated cellulose by P-3CR in DMSO, thus leading to samples with tunable thermal
93 properties (Söyler et al. 2018), and - 2,3-dialdehyde cellulose to form products with reduced Tg (Esen and
94 Meier 2020). In heterogeneous conditions, P-3CR was also carried out on tempo-oxidized cellulose
nanofibrils by Stenzel *et al.* to graft thermoresponsive polymer (Khine, Ganda, and Stenzel 2018) and bioactive moieties (Li *et al.* 2022), opening the way of promising new nanocellulose objects. Very recently, Hamzah *et al.* applied P-3CR to periodate oxidized microfibrillated cellulose in heterogeneous medium to anchor aliphatic chains at the surface (Hamzah *et al.* 2024). Our research team has devoted considerable efforts to the P-3CR modification of polysaccharides in water. We first proved the feasibility to Passerini modify CMC by a series of model aldehydes and isocyanides (Pettignano, Daunay, *et al.* 2019). The derivatives exhibit good adsorption ability onto cellulosic substrates. We were also interested in the functionalization of periodate oxidized nanofibrils of cellulose, to bring specific reactivities at the surface, further successfully exploited in aza-Michael reaction and copper-catalyzed Huisgen cycloaddition reactions (Pettignano *et al.* 2020). More recently, we have demonstrated the potential of α-substituted ketones, conjointly with isocyanide, to functionalize CMC via P-3CR, with the final goal to design thermosensitive chemical hydrogels (Remy *et al.* 2023).

With the aim of broadening the scope of original polysaccharide-based derivatives we present herein *i*) the Passerini dual hydrophobization of CMC and ALG in mild conditions, and *ii*) the potential of the modified derivatives to be processed as thin films. Indeed, while a substantial literature dealing with organization of hydrophobized polysaccharides in water exists, only few examples report on the development of films/coatings from modified polysaccharides (Bordenave, Grelier, and Coma 2010; Hartman, Albertsson, and Sjöberg 2006; Wang *et al.* 2020).

In this context, we make the hypothesis that the synthesis of never described CMC and ALG derivatives concomitantly modified by two hydrophobic moieties - allowed by Passerini reaction - will allow to engineer films exhibiting enhanced and adjustable wettability properties and hydration degrees. Thus, a particular attention is devoted to the effect of experimental parameters, and the versatility of the dual Passerini functionalization is addressed by using a large library of aldehydes and isocyanides which differ in their hydrophobicity and rigidity. Specifically, the reactivity of CMC into Passerini reaction is compared with the one of alginates of different molar masses. The modified polysaccharides were further processed as thin films and their wettability properties are discussed in link with the structure of the products. Finally, the film formation is carefully examined by QCM-D experiments coupled to AFM observations upon humid environment.

## 2. EXPERIMENTAL SECTION

### 2.1 Materials

Carboxymethyl cellulose sodium (CMC) and two alginate of low (ALG1) and medium viscosity (ALG2) were supplied by Sigma Aldrich. The degree of carboxymethylation of CMC given by the supplier is $DS_{COONa} = 1.20$ corresponding to an average number of 1.2 carboxymethyl groups per sugar unit. The commercially available CMC and ALG are naturally under the form of sodium carboxylate salts. The amount of -COONa groups of the two polysaccharides was experimentally confirmed by a conductometric titration of CMC and ALG aqueous solutions with an HCl solution (Figure S1). The equations 1 and 2 given
in SI part allowed to calculate the number of initial carboxylate groups, and values of 1.25 for CMC and 0.95 for ALG were found, that are very close to the values provided by the supplier. These polysaccharides have weight average molecular weights of 250 kg/mol, 46 kg/mol and 330 kg/mol, respectively as determined by Size Exclusion Chromatography. Moreover, the two ALG are characterized by a mannuronate/guluronate ratio (M/G) of 1.7, as calculated by ¹H NMR (Grasdalen, Larsen, and Smidsrød 1979). Butyraldehyde (-But-), heptaldehyde (-Hept-), undecanal (-Undeca-), 5-norbornene-2-carboxaldehyde (-Norb-), 10-undecenal (-Undece-), cyclohexyl isocyanide (-CH), tert-butyl isocyanide (-TertB), butyl isocyanide (-But), sodium chloride and poly(allylamine hydrochloride) (PAH) of 50 kg/mol were provided by Sigma Aldrich and were used without further purification. Acetone, isopropanol (IPA), dimethylsulfoxide (DMSO) and HCl (1M) were supplied by Carlo Erba.

## 2.2 General Procedure for the Passerini modification of CMC and ALG

### 2.2.1 Modification of CMC

A typical example is given for the sample 1 in Table 1. CMC (0.4 g, 1.55 mmol, 1 eq. COONa) was dissolved in 27 mL of deionized water to reach 1.5 w/v% solution. The solution was stirred overnight at 25 °C. To target a partial acidification at a DS<sub>COOH</sub> of 0.7 -COOH functional group per one sugar unit, 1M HCl was drop by drop introduced in the medium (1.08 mmol, 1.08 mL). By assuming its complete consumption, the pH of the solution was decreased from 7 down to 3.7. Under magnetic stirring, butyraldehyde (80 µL, 0.82 eq. relative to available COOH) and tert-butyl isocyanide (89 µL, 0.72 eq. relative to available COOH) were added dropwise in the reaction medium. The reaction was conducted for 18 h at 25 °C. The reaction medium was then precipitated into cold IPA (270 mL). The precipitate was filtrated on a fritted glass, and was washed four times in acetone (50 mL). The end-product was redissolved overnight in water and freeze-dried for 72 h. The products were stored in sealed vials and kept under inert gas. For sake of clarity, the abbreviations are indicated in Fig. 1.

The environment factor, defined as E-factor, is defined as the ratio of the loss material over the experimentally obtained product, according to Eq. (1):

$$E\text{-factor} = \frac{\text{weight of raw materials} - \text{weight of obtained product}}{\text{weight of obtained product}} \quad (1)$$

This factor metric does not take water and solvents into account, since this method was described as appropriated for early stages of development (Roschangar, Sheldon, and Senanayake 2015).

### 2.2.2 Modification of ALG

The Passerini reaction was then extended and adapted to ALG. A typical example of the synthesis is described for the sample 2 in Table 2.

ALG1 (0.4 g, 2.02 mmol, 1 eq. COONa) was dissolved in 27 mL of deionized water to reach 1.5 w/v% solution. The solution was stirred overnight at 25 °C. To target a partial acidification at a DS<sub>COOH</sub> of
0.7 -COOH function per one sugar unit, 1M HCl was drop by drop introduced in the medium (1.41 mmol, 1.41 mL). By assuming its complete consumption, the pH of the solution was decreased from 6.29 down to 2.56. Under magnetic stirring, butyraldehyde (105 µL, 0.82 eq. relative to available COOH) and tert-butyl isocyanide (117 µL, 0.72 eq. relative to available COOH) were added dropwise in the reaction medium. The reaction was carried out for 18 h at 25 °C. The reaction medium was then precipitated into cold IPA (270 mL). The precipitate was filtrated on a fritted glass, and was washed four times in acetone (50 mL). The end-product was redissolved overnight in water and freeze-dried for 72 h.

For ALG2, of higher viscosity, more diluted acidic solutions of 0.1M HCl were used in order to avoid the gelation of the medium induced by dimerization between the -COOH groups. The medium was left overnight, and any possible local gelation was efficiently redispersed during the stirring step.

## 2.3 Characterization of products

### 2.3.1 Infrared analysis (IR) spectroscopy

FTIR spectra were recorded using a Nicolet iS10 (ThermoFischer Scientific) in attenuated total reflectance mode (Smart iTR Diamond ATR), by an acquisition of 32 scans from 650 to 4000 cm⁻¹.

### 2.3.2 Nuclear magnetic resonance (NMR) spectroscopy

NMR spectra were recorded using a Bruker Avance 400 spectrometer. According to previous studies, the parameters are ¹H: 400 MHz, 512 scans, D1 = 10 s; ¹³C: 400 MHz, 12288 scans, D1 = 3 s; HSQC and HMBC: 400 MHz, 32 scans, D1 = 2 s. Spectra were mainly recorded by using 10 g/L solutions in D₂O at 298 K. However, if the solubility of the Passerini modified products in water is not complete, the NMR analysis were recorded in D₂O/organic solvent 50/50 v/v% mixture at 323 K. TSP-d4 was used as an internal reference.

The values of degree of substitution of the Passerini modified polysaccharides, referred as DS, correspond to the average number of grafted α-acyloxycarboxamide function per one sugar unit of CMC or ALG. DS was calculated from the ¹H NMR spectra by using Eq. (2) and Eq. (3).

$$m_{\text{graft}} = \frac{m_{\text{TSP-d4}}}{M_{\text{TSP-d4}}} \times \frac{H_{\text{TSP-d4}}}{I_{\text{TSP-d4}}} \times \frac{I_{\text{graft}}}{H_{\text{graft}}} \times M_{\text{graft}} \quad (2)$$

$$DS = \frac{m_{\text{graft}}/M_{\text{graft}}}{(m_{\text{total}} - m_{\text{graft}})/M_{\text{sugar unit}}} \quad (3)$$

where mTSP-d4 refers to the mass of the internal reference TSP-d4, mtotal to the mass of the total polymer dissolved in the NMR tube and mgraft to the mass of the graft. Igraft and ITSP-d4 correspond to the integration of the well-defined peaks assigned to a chemical group of the grafted moieties and to the internal reference (TSP-d4), respectively. HTSP-d4 and Hgraft correspond to the number of protons corresponding to the integrated peaks. M values refer to the molar masses of TSP-d4, to the one of one sugar unit, and to the one of the covalently grafted residue (Mgraft = Mw(aldehyde) + Mw(isocyanide) +1).
By using the DS (Eq. 3), we can calculate the mass of expected product $m_{theo}$, calculated through Eq. 4-5, by taking into account the change in molar mass resulting from the Passerini reaction. The mass yield is then obtained through Eq. 6 by dividing the mass of the recovered product $m_{exp}$ by the one of the expected one $m_{theo}$.

$$m_{theo} = \frac{m_{init} * M_{P3CR}}{M_{sugar unit}} \quad (4)$$

$$M_{P3CR} = M_{sugar unit} + DS \times M_{graft} \quad (5)$$

$$\text{mass yield} = \frac{m_{exp}}{m_{theo}} \times 100 \% \quad (6)$$

Where $m_{init}$ and $M_{sugar unit}$ refer to the mass of the polysaccharide initially weighed, and to the molar mass of the corresponding sugar unit, respectively.

### 2.3.3 Size exclusion chromatography (SEC)

The analysis was conducted realized using a chromatograph equipped with two PL aquagel OH columns (mixed-M and mixed-H, 8 µm) coupled with a combination of detectors (Light Scattering Instrument: HELEOS II and Refractive Index: Optilab rEX). The eluent was a 0.1 M $NaNO_3$ solution, adjusted to pH 7 with a NaOH solution flowing at 0.5 mL/min. Polysaccharide solutions were prepared in $NaNO_3$ solution at a concentration of 1 mg/mL, and filtered through a CME 0.45 µm syringe filter.

## 2.4 Characterization wettability of thin films

### 2.4.1 Polysaccharide film preparation

Si wafers (100 mm, P-type, Boron-doped, <100>) were cut into 2 cm x 2 cm squares, exhaustively rinsed with Milli-Q water and ethanol, dried under nitrogen gas and finally cleaned by UV-Ozone treatment for 30 min (UVO-CLEANER® Model 42 SERIES). Polysaccharide solutions were prepared at 1 g/L in milliQ water and filtered with a 0.8 µm cellulose filter. Spin-coating was undertaken on a POLOS200 Spin Coater. Three drops of the solution were deposited onto a cleaned Si surface and were spin-coated at 2000 rpm for 3 min. The deposition was repeated five times. The spin-coated silicon wafers were then dried overnight in an oven at 60 °C. The thicknesses of the films, at dry state, were determined by using a reflectance analyzer (Filmetrics, F20-UV model) connected to the FILMeasure software.

### 2.4.2 Analysis of surface topography by AFM

#### AFM in Tapping mode

AFM images were acquired in Tapping mode, onto silicon wafers with a size of 1 cm × 1 cm, in air at room temperature using an AFM Bruker Multimode 8 apparatus equipped with Nanoscope V controller. The cantilevers were purchased from Bruker with the model of Bruker RTESP-150. To access to the film thickness at dry state, an indentation was performed with a razor blade to detect the silicon wafer and to
222

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eliminate the removed polymer from the substrate. The height difference observed after the indentation is probed by AFM in topology mode, which gives the film thickness. Five measurements were carried on each sample to compute an average thickness value.

225

### AFM in PeakForce (PF) Tapping mode

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230

To observe swollen polysaccharide thin films at high resolution and in operando, we used an AFM working in liquid, directly on QCM-D sensors, carefully rinsed with water and dried. Images were acquired by using an AFM Bruker Dimension Icon with Nanoscope 6 controller. The imaging conditions used were a PF frequency of 2 KHz and apply forces between 450 pN and 750 pN. The cantilevers were purchased from Bruker with the model of Bruker ScanAsyst-Fluid (k = 0.7 N/m) adapted to observations in liquids.

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### 2.4.3 Measurements of water contact angles

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Water contact angles (WCA) were measured on the spin-coated surfaces by using a tensiometer Dataphysics OCA25. Each experiment was reproduced for at least 5 drops of 2 µL of Milli-Q water and contact angles were considered 10 s after the deposition. It ensured the analysis of stable drops, and reproducibility of data. At least five angle determination at different locations of the surface were done to get an average value with a standard deviation. All the measurements were taken at T = 18.4 °C ± 1.1 °C and RH = 30.5 % ± 3.0.

238

## 2.5 Quartz Crystal Microbalance with Energy Dissipation Monitoring (QCM-D)

239

### 2.5.1 Film preparation

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255

QCM-D experiments were performed with a Q-Sense Explorer device. The commercially available QCM-D quartz crystals were covered with gold electrodes (QSX 301, Q-Sense, Sweden) and have a fundamental resonance frequency ($f_0$) of 5 MHz. The temperature of the analysis chamber was fixed at 20 °C. The gold QCM-D-sensor were prealably rinsed with MilliQ water and ethanol and further dried with nitrogen gas. The polymers (unmodified CMC/ALG and Passerini modified CMC/ALG) were dissolved at 1.5 g/L in 0.1M NaCl solution and filtered onto a 0.8 µm cellulose filter (ROTILABO® CME). To start the QCM-D analysis, a first stabilization step was carried out by injecting a solution of NaCl at 0.1M for 30 min at a flow rate of 100 µL.min$^{-1}$, allowing to reach the equilibrium (i.e. when normalized frequency $\Delta f_n/n$ and dissipation $\Delta D_n$ responses give stable baselines)). After this stabilization step, a solution of poly(allyl)amine (PAH: polycation) (1.5 g/L in NaCl 0.1 M) was firstly injected. PAH acts as an anchoring polymer to favor the adsorption of the polyanion CMC (or ALG) onto the electrode sensor. The PAH injection was followed by a rinsing step by flushing a 0.1M NaCl solution. Then, the solution of polysaccharide derivative was flowed onto the surface to allow the adsorption until to reach the equilibrium state, followed by a rinsing step by flushing the chamber with a 0.1M NaCl solution, in order to remove the polymer chains weakly bound to the surface. This step allows to examine the reversibility of the adsorption.
Considering that a polymer forms a thin and rigid film (relatively low $\Delta D_n/n$, and good superimposition of all harmonics) onto the surface of the sensor upon adsorption, $\Delta f_n/n$ is related to the adsorbed mass per unit area $\Delta m$ through the Sauerbrey equation, Eq. (7).

where $n$ is the number of the overtone, and $C$ is the mass sensitivity constant which depends on the material properties of the quartz and its fundamental resonance frequency (Sauerbrey 1991)

$$ \Delta m = -C \times \Delta f_n/n \qquad (7) $$
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Aldehyde R₁ =</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Isocyanide R₂ =</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 1.** Partial acidification of (A) CMC and (B) ALG and subsequent Passerini three-component reaction with hydrophobic aldehydes (R₁) and isocyanides (R₂). Alginate is composed of GG, MM, and MG sequences, but for simplicity, only GG and MM are represented.</figcaption></figure>
<table><caption>**Table 1.** P-3CR reactions applied to CMC with various aldehydes and isocyanides: experimental conditions and reaction characteristics</caption><thead><tr><th>Entry</th><th>Sample<sup>a</sup></th><th>Eq COOH</th><th>Eq. R<sub>1</sub><sup>b</sup></th><th>Eq. R<sub>2</sub><sup>b</sup></th><th>Solvent</th><th>DS (± 5 %)<sup>c</sup></th><th>Graft. Eff R<sub>2</sub> (%)<sup>d</sup></th><th>Graft. Eff COOH (%)<sup>e</sup></th><th>Mass yield (%)<sup>f</sup></th><th>E-factor<sup>g</sup></th></tr></thead><tbody><tr><td>1</td><td>CMC-But-TertB</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.31/0.35/0.33/0.36/0.43/49/46/50/43/0.33/0.31</td><td>6/46/43</td><td>44/50/47/51/47/47/44</td><td>86/90/70/87/n.d./87/99</td><td>4.5/4.1/3.6/3.3/4.4/3.8</td></tr><tr><td>2</td><td>-</td><td>0.70</td><td>0.41</td><td>0.36</td><td>H<sub>2</sub>O</td><td>0.19/0.13</td><td>53/36</td><td>27/18</td><td>89/89</td><td>4.4/4.7</td></tr><tr><td>3</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.44</td><td>15</td><td>63</td><td>80</td><td>n.a.</td></tr><tr><td>4</td><td>CMC-But-CH</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.23</td><td>32</td><td>33</td><td>84</td><td>4.9</td></tr><tr><td>5</td><td>CMC-But-ButN</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.35/0.33</td><td>49/46</td><td>50/47</td><td>90/78</td><td>3.9/5.0</td></tr><tr><td>6</td><td>CMC-Hept TertB</td><td>0.70</td><td>0.41</td><td>0.36</td><td>H<sub>2</sub>O</td><td>0.06</td><td>17</td><td>8</td><td>94</td><td>4.6</td></tr><tr><td>7</td><td>-</td><td>0.70</td><td>0.62</td><td>0.54</td><td>H<sub>2</sub>O</td><td>0.09</td><td>17</td><td>13</td><td>94</td><td>4.6</td></tr><tr><td>8</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.10*/0.13*</td><td>14/18</td><td>14/18</td><td>n.a./84/100</td><td>n.a./4.2</td></tr><tr><td>9</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.25*</td><td>9/13</td><td>36/54</td><td>n.a./n.a.</td><td>n.a.</td></tr><tr><td>10</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O/IPA<sup>h</sup></td><td>0.12</td><td>17</td><td>17</td><td>77</td><td>5.8</td></tr><tr><td>11</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O/DMSO<sup>h</sup></td><td>0.12</td><td>17</td><td>17</td><td>78</td><td>5.8</td></tr><tr><td>12</td><td>CMC-Undeca-TertB</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0</td><td>0</td><td>0</td><td>95</td><td>5.2</td></tr><tr><td>13</td><td>-</td><td>0.70</td><td>1.65</td><td>1.44</td><td>H<sub>2</sub>O</td><td>0</td><td>0</td><td>0</td><td>90</td><td>6.2</td></tr><tr><td>14</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O/IPA<sup>h</sup></td><td>0.10</td><td>14</td><td>14</td><td>80</td><td>5.7</td></tr><tr><td>15</td><td>CMC-Undeca-TertB</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0</td><td>0</td><td>0</td><td>92</td><td>5.2</td></tr><tr><td>16</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O/IPA<sup>h</sup></td><td>0.17/0.17</td><td>24/24</td><td>24/24</td><td>83/70</td><td>5.0/5.5</td></tr><tr><td>17</td><td>-</td><td>0.70</td><td>3.88</td><td>2.88</td><td>H<sub>2</sub>O/IPA<sup>h</sup></td><td>0.39</td><td>14</td><td>56</td><td>n.d.</td><td>n.a.</td></tr><tr><td>18</td><td>CMC-Norb-TertB</td><td>0.70</td><td>0.62</td><td>0.54</td><td>H<sub>2</sub>O</td><td>0.24*</td><td>44</td><td>34</td><td>85</td><td>4.2</td></tr><tr><td>19</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.34*/0.33*</td><td>47/46</td><td>48/47</td><td>n.a./95</td><td>n.a./3.8</td></tr><tr><td>20</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.34*/0.44*</td><td>12/15</td><td>48/63</td><td>68/ n.a.</td><td>7.3/n.a.</td></tr></tbody></table>
<table><tr><td>21</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O/DMSO<sup>h</sup></td><td>0.25</td><td>35</td><td>36</td><td>98</td><td>3.9</td></tr><tr><td>22</td><td>CMC-Norb-ButN</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.12</td><td>17</td><td>17</td><td>94</td><td>n.a.</td></tr></table>

a Reaction conditions: 1.5 w/v% of CMC, 18-20 h, 25 °C b Equivalent of reagents with respect to available COOH functions. c DS: degree of substitution, number of grafted substituents per sugar unit, calculated by ¹H NMR (see Experimental section). d Grafting efficiency R₂ (%) = obtained DS / equiv of employed R₂. e Grafting efficiency COOH (%) = obtained DS / DS COOH, f Mass yield, g E-factor, h Dissolution in 50/50 v/v% water/co-solvent mixture * thickening/gelation of the medium during the reaction. n.a.: non-applicable.

<table><caption>**Table 2.** P-3CR applied to ALG with various aldehydes and tert-butyl isocyanide: experimental conditions and reaction characteristics</caption><thead><tr><th>Entry</th><th>Sample<sup>a</sup></th><th>Eq. COOH</th><th>Eq. R<sub>1</sub><sup>b</sup></th><th>Eq. R<sub>2</sub><sup>b</sup></th><th>Solvent</th><th>DS (± 5 %)<sup>c</sup></th><th>Graft. Eff R<sub>2</sub> (%)<sup>d</sup></th><th>Graft. Eff COOH (%)<sup>e</sup></th><th>Mass yield (%)<sup>f</sup></th><th>E-factor<sup>g</sup></th></tr></thead><tbody><tr><td>1</td><td>ALG1-But-TertB</td><td>0.70</td><td>0.62</td><td>0.54</td><td>H<sub>2</sub>O</td><td>0.21/0.21</td><td>39/39</td><td>30/30</td><td>67/60</td><td>8.0/8.8</td></tr><tr><td>2</td><td>-</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.40/0.43/0.44</td><td>55/60/61</td><td>57/61/63</td><td>79/82/ n.a.</td><td>5.9/4.9/n.a.</td></tr><tr><td>3</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.44</td><td>15</td><td>63</td><td>60</td><td>9.3</td></tr><tr><td>4</td><td>ALG1-Hept-TertB</td><td>0.58</td><td>0.62</td><td>0.54</td><td>H<sub>2</sub>O</td><td>0.11</td><td>20</td><td>19</td><td>n.a.</td><td>n.a.</td></tr><tr><td>5</td><td>-</td><td>0.58</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.16</td><td>22</td><td>28</td><td>98</td><td>4.6</td></tr><tr><td>6</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.41**/0.46**</td><td>14/16</td><td>58/65</td><td>61/ n.a.</td><td>8.7/n.a.</td></tr><tr><td>7</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O/IPA<sup>h</sup></td><td>0.03</td><td>4</td><td>1</td><td>46</td><td>n.a</td></tr><tr><td>8</td><td>ALG1-Norb-TertB</td><td>0.58</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.38</td><td>53</td><td>66</td><td>67</td><td>5.9</td></tr><tr><td>9</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.34</td><td>12</td><td>48</td><td>n.a.</td><td>8.7</td></tr><tr><td>10</td><td>ALG1-Undece-TertB</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O/IPA<sup>h</sup></td><td>0.02</td><td>3</td><td>1</td><td>16</td><td>n.a</td></tr><tr><td>11</td><td>ALG2-But-TertB</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.22</td><td>30</td><td>31</td><td>92</td><td>5.5</td></tr><tr><td>12</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.42/0.5</td><td>14/17</td><td>60/71</td><td>77/ n.a.</td><td>6.7/n.a.</td></tr><tr><td>13</td><td>ALG2-Hept-TertB</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.07</td><td>10</td><td>10</td><td>77</td><td>7.8</td></tr><tr><td>14</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.23*</td><td>8</td><td>33</td><td>62</td><td>10.7</td></tr></tbody></table>
<table><tr><td>15</td><td>ALG2-Norb-TertB</td><td>0.70</td><td>0.82</td><td>0.72</td><td>H<sub>2</sub>O</td><td>0.13</td><td>18</td><td>18</td><td>79</td><td>7.1</td></tr><tr><td>16</td><td>-</td><td>0.70</td><td>3.28</td><td>2.88</td><td>H<sub>2</sub>O</td><td>0.25*</td><td>10</td><td>36</td><td>n.a.</td><td>n.a.</td></tr></table>

a Reaction conditions: 1.5 w/v% of ALG1 and 1 w/v% of ALG2, 18-20h, 25 °C b Equivalent of reagents with respect to available COOH functions. c DS: degree of substitution, number of grafted substituents per sugar unit, calculated by ¹H NMR (see Experimental section). d Grafting efficiency R₂ (%) = obtained DS / equiv of employed R₂, e Grafting efficiency COOH

(%) = obtained DS / DS COOH , f Mass yield, g E-factor, h Dissolution in 50/50 v/v% water/co-solvent mixture,

* thickening/gelation of the medium during the reaction. ** precipitation of the polysaccharide during the reaction. n.a.: non-applicable.
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## 3. RESULTS AND DISCUSSION

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### 3.1 Dual hydrophobization of polysaccharides by P-3CR.

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As previously reported, CMC can be successfully modified by P-3CR in aqueous medium, by using model isocyanides and aldehydes (Pettignano, Daunay, et al. 2019). Herein, we aim a dual hydrophobization of carboxylated polysaccharides, i.e. CMC (250 kg/mol) and alginate (ALG) of various molar masses (ALG1 = 46 kg/mol and ALG2 = 330 kg/mol), to further processed them as thin films. As mentioned, ALG has never been modified by P-3CR so far. Thus, aliphatic isocyanides, such as cyclohexyl (-CH), tert-butyl (-TertB) and butyl (-ButN) isocyanide were employed, conjointly with a series of fatty aldehydes containing aliphatic chains (saturated and unsaturated) which differ in their hydrophobicity through their chain length: butyraldehyde (-But), heptaldehyde (-Hept-), undecanal (-Undeca-), 10-undecenal (-Undece-) and a bulky aldehyde: 5-norbornene-2-carboxaldehyde (-Norb-) (Fig.1, Table 1). The objective is twofold: - generating a panel of never described dually hydrophobized polysaccharide derivatives and - comparing CMC with ALG which completely differ in their structures in terms of chain conformation, nature of sugar units, pKa value, number and distribution of carboxy groups.

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Both ALG1 and ALG2 exhibit a M/G ratio of 1.7, as calculated from NMR ¹H analysis (Grasdalen et al. 1979). To engage the Passerini reaction, we adopted the process conditions to our previous work that allowed to identify optimized experimental parameters (Pettignano, Daunay, et al. 2019). In particular, a sufficient number of carboxylic acid groups -COOH involved into the Passerini reaction must be generated, while guarantying the water solubility of the partially acidified CMC by avoiding the dimerization between -COOH.

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Thus, P-3CR with CMC were carried out from a solution at pH = 3.70, that allowed to acidify 58 % of -COONa and to provide 0.7 -COOH functions per sugar unit. In the case of ALG, in order to obtain the same number of -COOH functions per sugar unit, 70 % of -COONa groups were acidified, thus yielding a pH of 2.5. This noticeably lower pH can be attributed to the lower pKa values of ALG (at 3.65 and 3.38) (Vleugels et al. 2018) and to the lower concentration of remaining carboxylate residues after acidification because alginate chains carry one carboxylate group per sugar unit, whereas CMC chains bear in average 1.2 carboxylate groups per unit. Such a low pH led to a partial gelation of the medium for the highest M<sub>w</sub> of alginate, which required a stirring stage overnight. As previously proved in the case of CMC (Pettignano, Daunay, et al. 2019), SEC analysis shown the integrity of alginate chains during the acidification step (Figure S2). The partial acidification of alginate was confirmed by IR spectroscopy and a typical example is given for ALG1 (Fig. 2A and 2B). A peak at 1740 cm<sup>-1</sup> appears and is attributed to the stretching of the -C=O of -COOH groups, while bands at 1410 and 1600 cm<sup>-1</sup> correspond to the remaining -COO<sup>-</sup> groups.
<figure><img src="image_12.png" /><figcaption>**Fig. 2.** ATR-IR of (A) ALG1, (B) Partially protonated ALG1, and (C) Passerini product ALG1-But-*Tert*B (Table 2, Entry 2)</figcaption></figure>

The acidification step was followed by the addition of the aldehyde and isocyanide into the medium. The reactions were then carried overnight at 25 °C, and the products were purified by precipitation in IPA followed by four successive washings in acetone, leading to the recovering of pure product in very good mass yields (60 up to around 100 %w/w).

By taking the sample ALG1-But<sub>0.82</sub>-*Tert*B<sub>0.72</sub>, (Table 2, entry 2) as an example, additional IR bands at 1660 and 1540 cm<sup>-1</sup> attributed to amide I and amide II signals, respectively, emerge which confirms the formation of the α-acyloxycarboxamide function by P-3CR (Fig. 2C). Moreover, the grafting is visible through the more pronounced C-H stretching band at 2800-3000 cm<sup>-1</sup> in the IR spectrum C in Figure 2. Also, IR shows the absence of residual isocyanide around 2250 cm<sup>-1</sup>, attributed to the elongation of -C≡N bonds, proving the efficiency of the purification process. The products were in-depth characterized by <sup>1</sup>H and <sup>13</sup>C NMR spectroscopy and by 2D NMR experiments. As examples, the <sup>1</sup>H and <sup>13</sup>C NMR spectra of ALG1-But-*Tert*B (Table 2, Entry 2) are shown in Figure 3A and 3B.

<figure><img src="image_13.png" /><figcaption>**Fig. 3.** A)<sup>1</sup>H NMR spectrum (D<sub>2</sub>O at 298K) and B)<sup>13</sup>C NMR spectrum (D<sub>2</sub>O/Acetonitrile-d3 50/50 v/v% at 323K) of ALG1-But-*Tert*B (Table 2, Entry 2).</figcaption></figure>

In the <sup>1</sup>H spectrum, protons of alginate appear in the region between 3.4 and 5.2 ppm. The Passerini ligation leads to the apparition of new signals related to the protons of the grafted butyl (2 protons at 1.74 ppm, 2 protons at 1.36 ppm, 3 protons at 0.91 ppm), and *tert*-butyl (9 equivalent protons at 13, 14, 15).
1.29 ppm) moieties (Figure 3A). The chemical grafting is also evidenced in the ¹³C NMR spectrum with the emergence of the carbon signals of the butyl (carbon 8 at 33 ppm, carbon 9 at 17.6 ppm, carbon 10 at 12.9 ppm) and the *tert*-butyl (carbon 12 at 51.2 ppm and carbons 13-15 at 27.7 ppm) substituents (Figure 3B). The signals of carbons 6 and 11 at 168.5 ppm and 169.8 ppm correspond to the carbonyl function of the ester and the amide bonds of α-acyloxycarboxamide, respectively. The carbons of the residual carboxylate salts and carboxylic acids carbon 6' and 6" are visible at 173 ppm. 2D NMR experiments allowed to confirm the peak attribution. HSQC was used to uncover proton-carbon single bond correlations (¹J) (Figure S3), whereas HMBC probes the long-range correlations between carbons and protons (²J-³J) (Figure 4).

<figure><img src="image_14.png" /><figcaption>**Fig. 4.** HMBC NMR spectrum of ALG1-But-*Tert*B (Table 2, Entry 2), recorded in D₂O/Acetonitrile-d3 50/50 v/v% at 323K.</figcaption></figure>

HMBC also provided unambiguous evidence of the Passerini reaction through the presence of the cross-peaks between the proton at 5.46 ppm and the carbons of the carbonyls at 168.5 and 169.8 ppm which prove the scalar coupling in ³J *i*) between the proton 7 and the carbon 6 of the ester bond and *ii*) between the proton 7 and the carbon 11 of the amide bond (Figure 4). Such correlations confirm the covalent ligation through the α-acyloxycarboxamide link. The ¹H NMR analysis was then exploited to quantify the degree of substitution (*DS*) after Passerini reaction, by using TSP-d4 as an internal standard (see experimental section). In the case of ALG1-But-TertB (Table 2, Entry 2), the *DS* is around 0.40, which is higher than the value obtained with CMC (Table 1) and with ALG2 (Table 2) (explanations *vide-infra*). IR and ¹H NMR spectra of all products are available in Fig. S3-13.
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A large number of Passerini reactions were carried out on CMC, ALG1, and ALG2 (Tables 1 and 2), and for all experiments the aldehyde/isocyanide ratio was kept constant at 1.14 (a slight excess of aldehyde with respect to isocyanide was chosen to favor the bound intermediate allowing the activation of the oxo-compound, that was further attacked by the isocyanide). The corresponding IR and NMR spectra of all modified products are given in Figures S4-S14.

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We were firstly interested in extending the acquired knowledge on Passerini functionalization of CMC to the grafting of other types of hydrophobic substituents (Table 1) (Pettignano, Daunay, et al. 2019; Remy et al. 2023). The reproductibility of the Passerini of CMC with butyraldehyde (-But) and *tert*-butyl isocyanide (*-TertB*) was firstly proved by undertaking successive synthesis in similar experimental conditions, which all provided DS values at the same order of magnitude (Table 1, entry 1). We then varied the amount of reactants in the case of CMC-But-*TertB* (Table 1, entries 1-3), and it results that the increase of the quantity of reactants leads to an increase of DS ranging from 0.13 up to 0.44 (corresponding to a COOH conversion of 63 %).

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The impact of the structure of the isocyanides was then considered. By using a well water-soluble aldehyde, *i.e.* only butyraldehyde, upon the same experimental conditions (Entries 1, 3 and 5), similar values of DS (around 0.33) were obtained for *tert*-butyl isocyanide and butyl isocyanide and the values are higher than the one obtained with cyclohexyl isocyanide (DS = 0.23, Table 1, entry 4). The lower reactivity of cyclohexyl isocyanide can be allocated to its limited water solubility and the steric hindrance caused by the cycloaliphatic ring. Note that this dual hydrophobization leads to samples with decreased water solubility, but maintained up to 50 g/L.

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Thus, by using *tert*-butyl isocyanide, we examined the impact of the structure of the aldehyde, by employing *i*) saturated fatty aldehydes with various chain lengths: in C4 (-But- Entries 1-5), as previously discussed, in C7 (-Hept- Entries 6-11) and in C11 (-Undeca- Entries 12-14), *ii*) unsaturated chains in C11 (-Undece-, Entries 15-17) and *iii*) bulky moieties (-Norb-, entries 18-22). As expected, the reactivity of the fatty aldehydes in aqueous medium is decreased with the increase of the chain length. For the chain in C7, the reaction (Table 1, entries 6-9) leads to DS varying from 0.06 up to 0.25 (against 0.44 for the C4). This lower reactivity is due to the increase of the molar mass of the modifier and by its reinforced hydrophobicity (the C7 molecules becomes insoluble in water for C > 1.25 g/L). For entry 9, corresponding to higher amount of reactants, the reaction medium is prone to an increase in viscosity. This result emphasizes that a suited balance between the extent of the grafting and the water solubility of the modified product should be ensured. P-3CR with longer fatty aldehydes, in C11 (saturated or unsaturated one, Table 1, entries 12-13 and 15), was not conclusive due to their insolubility in water. Given these solubility issues, P3CR with long fatty aldehydes was attempted in mixtures of miscible co-solvents: (H₂O (60 %) / IPA (40 %), and H₂O (60 %) / DMSO (40 %)) (Table 1, entries 14, 16-17). The solvent proportions were chosen to ensure the solubility of the reactants, and to keep the solubility of the partially protonated CMC in these conditions (C = 15 g/L in the solvent mixture). The use of a co-solvent allowed to decrease the viscosity of the reaction medium. However, as shown by the entries 10-11 (table 1), the addition of IPA or DMSO in the case of aldehyde in C7 did not allow to improve the grafting efficiency (DS = 0.12). Regarding the use of -undeca- and -undece- (entries 14 and 16-17, Table 1), the addition of IPA favored the Passerini coupling and DS of 0.10 and 0.17 were obtained, respectively. A bulky aldehyde, 5-norbornene-2-carboxaldehyde, was finally

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employed, conjointly with *tert*-butyl isocyanide (entries 18-21, Table 1) and butyl isocyanide (entry 22, Table 1). This norbornene-based compound is of particular interest, as not only it is hydrophobic but the double bond is known to be reactive upon photo-induced thiol-ene reactions, which paves the way for further post reactions (Lin, Ki, and Shih 2015). The grafting of 5-norbornene-2-carboxaldehyde with the *tert*-butyl aldehyde leads to interesting DS values around 0.34, which is higher than for butyl isocyanide (Table 1, entry 22, DS = 0.12). When norbornene is employed, the macroscopical aspect of the reaction medium evolves during the reaction and a substantial increase in viscosity is observed. We can reasonably do the hypothesis that such an increase in viscosity results from the formation of interchain hydrophobic interactions between the grafted norbornene resides. A threshold value of DS = 0.34 seems to be reached, probably due to the sterical hindrance brought by the bulky norbornene moiety. Moreover, the addition of DMSO (entry 21, Table 1) allows to diminish the viscosity of the medium by most probably avoiding the formation of norb/norb interactions but does not contribute to increase the DS, as previously shown for Passerini reaction of CMC with ketones and isocyanides (Remy et al. 2023).

Interestingly, we aimed at extending the Passerini reaction to alginate. Two different alginates (ALG1: 46 kg/mol, and ALG2: 330 kg/mol) were subjected to Passerini by using butyraldehyde (-But-), heptaldehyde (-Hept-), 10-undecenal (-Undece-) or 5-norbornene-2-carboxaldehyde (-Norb-) conjointly with *tert*-butyl isocyanide (*-TertB*), which was shown to be the more reactive isocyanide (Table 1). As previously mentioned, in spite of the attractive presence of carboxy groups, ALG has never been modified by Passerini reaction so far. Globally, Table 2 shows a good reactivity of ALG1. Under similar experimental conditions, ALG1-But-*TertB* with DS ranging from 0.21 to 0.44, ALG1-Hept-*TertB* with DS varying from 0.11 to 0.46, and ALG1-Norb-*TertB* with DS around 0.35 were generated in water (giving a COOH conversion of 63, 63 and 50 %, respectively). As for CMC, the reactivity of heptaldehyde is lower than the one of butyraldehyde (DS = 0.11 entry 4 against 0.21 entry 1 or 0.16 entry 5 against 0.4 entry 2) linked to the solubility issues. The use of an excess of reagents of Hept and *TertB* (entry 6) leads to the *in situ* precipitation of the modified polymer during the reaction, due to the presence of the apolar substituents. The grafting of Norb with *TertB* is also more efficient in the case of ALG1, compared to CMC (entries 8 and 9). No viscosity increase was observed, which indirectly shows that the molar mass of the polysaccharide plays a key role on the behavior of the modified product in water. IPA was then employed as a co-solvent, and contrary to the CMC modification, IPA is detrimental and did not favor the grafting of heptaldehyde and 10-undecenal onto ALG1, which is imputed to a lack of solubility of the partially acidified alginate chains in the IPA/H₂O mixture (Table 2, entries 7 and 10). Globally, the Passerini grafting efficiencies obtained with ALG1 are higher compared to CMC. The distribution and the number of the -COOH groups along the partially protonated alginate chains differ from the one of CMC. Regarding ALG, there is 1 COOH group per sugar unit against 1.2 in average in the case of CMC for which it is possible to have multiple neighboring COOH groups on the same sugar, thus leading to sterical hindrance and altering the reactivity. Moreover, the COOH groups of ALG might be less involved into the formation of H-bonds (COOH self-association or/and H-bonds with OH groups) compared to CMC, making them more accessible and more available. Finally, ALG1 having a molar mass well lower than the one of CMC, leads to a lower viscosity in water, thus facilitating the stirring and the chemical ligation. Then, we were interested in modifying ALG2 with higher molecular weight (330 kg/mol) and the same M/G ratio than
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ALG1. The table 2 (entries 10-15) shows that the reactivity of ALG2 is weaker, compared to ALG1, due to its higher molar mass which induces a higher viscosity. Increasing time or/and temperature should result in higher DS, but a long reaction time in water and a high temperature might induce a partial hydrolysis of the formed ester bond.

As the initial COONa groups of CMC and ALG undergo a protonation during the acidification step and are subsequently consumed during the Passerini reaction, the *in situ* monitoring of the pH of the medium enables the tracking of the reaction kinetics (Figure 5).

<figure><img src="image_15.png" /></figure>

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**Fig. 5.** Evolution of pH of CMC (line), ALG1 (dashed line) and ALG2 (dotted line) during the partial acidification step and the Passerini reaction with 0.82 eq. of butyraldehyde and 0.72 eq. of *tert*-butyl isocyanide (Entry 3, Table 1, Entry 2, Table 2, Entry 11, Table 2)

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The solubilization of CMC and ALG in pure water gives a pH of 6.95 and 6.28, respectively and the addition of an appropriated HCl volume yields a sharp pH drop down to 3.65 for CMC and 2.55 for ALG. The addition of the reagents leads to the consumption of the *in situ* generated -COOH groups by P-3CR. After a given time, the pH reaches a plateau (after 1 h for ALG1 and ALG2 and 5 h for CMC), highlighting the completion of the reaction. The reactions are particularly fast, contrary to more commonly used reactions to modify polysaccharides and under similar experimental conditions, the kinetics is much higher in the case of ALG. Given that the ester group present in the α-acyloxycarboxamide function, resulting from P-3CR, is sensitive to hydrolysis, the stability of the products in water at different temperatures was examined by ¹H NMR. The samples were equilibrated at the selected temperature and ¹H NMR spectra were recorded every 60 min for 375 min (Fig. S15-18). The analysis was carried out on CMC-But-*Tert*B DS = 0.34 (Table 1, entry 1), CMC-Norb-*Tert*B DS = 0.34 (Table 1, entry 19), ALG1-Norb-*Tert*B DS = 0.34 (Table 2, entry 9) and ALG2-Norb-*Tert*B DS = 0.25 (Table 2, entry 16) at 25 °C, 50 °C and 70 °C (Figure 6A and 6B).
<figure><img src="image_16.png" /><figcaption>**Fig. 6.** Evolution of hydrolysis rate of A) CMC derivatives and B) ALG1 derivatives, recorded every 60 min at 25, 50 and 70 °C.</figcaption></figure>

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Hydrolysis rates were determined from the integration of peaks resulting from ester hydrolysis, that are well identified by NMR spectroscopy. As a typical example, hydrolysis of CMC-But-TertB was quantified through the disappearance of the signal of proton 9 at 4.94 ppm at 25, 50 and 70 °C (Fig. S15). As depicted in Fig. 6A and 6B, whatever the structure of the polysaccharide derivative, hydrolysis is promoted by temperature, in agreement with previous results on CMC modified by P-3CR with ketones and isocyanides (Remy et al. 2023) and is quasi negligible at 25°C (around 3 %). The impact of the hydrophobic grafted structure was then considered by comparing CMC-But-TertB and CMC-Norb-TertB with similar DS (Fig. 6A and Fig. S16). CMC-Norb-TertB exhibits a reduced hydrolysis sensitivity, with a rate reaching 12 % at 70 °C and 6 % at 50 °C, against 17 % at 70 °C, 7 % at 50 °C for CMC-But-TertB. The bulky norbornene hydrophobic aldehyde acts as a shield enhancing the stability against hydrolysis. The behavior of the Passerini-modified alginates, i.e. ALG1-Norb-TertB and ALG2-Norb-TertB was also scrutinized (details in Fig. S17 and S18). At 70 °C, the hydrolysis rates of the two samples are similar and attain 31 %, but at 50 °C the rate drops down to 12 % to become negligible at 25 °C (around 2-3%). The higher sensitivity of ALG derivatives can be allocated to the distribution of -COOH groups along the chains, making α-acyloxycarboxamide bonds probably more exposed to water. Finally, the *E*-factors of each synthesis gathered in Tables 1 and 2 were calculated from the equation 1 (experimental part). This metric gives a good estimation of the amount of waste generated during the chemical processes, and has been employed to compare different synthetic pathways used to modify cellulose (Onwukamike *et al.* 2019). Herein, the *E*-factors are relatively low ranging from 3.3 to 10.7, which emphasizes the sustainability of the Passerini reaction in terms of atom economy and low waste generation.

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## 3.2 Properties of Passerini-modified polysaccharides films.

The hydrophobization of polysaccharides is of great interest for various applications both at liquid and solid state. A previous work of Pettignano *et al.* allowed to underscore the potential of using Passerini
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modified polysaccharides as thin films. Herein, in order to quantify the impact of the double hydrophobic modification, the derivatives were deposited onto silicon wafers and water contact angles (WCA) were measured after a settled time. The deposition was optimized to obtain well-covering films and films of 60 nm and 20 nm were obtained from CMC- and ALG1-based derivatives, respectively. The resulting WCA are shown in Figure 7, and allowed to elucidate the effect of i) the structure of the grafted hydrophobic moieties, ii) the extents of modification and iii) the nature of the polysaccharide.

<figure><img src="image_17.png" /></figure>

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**Fig. 7.** Average water contact angles (WCA) of CMC- and ALG1-based films determined 10 s after deposition of water droplets, at 25 °C. Pictures of deposited water drop and AFM height images in Tapping mode (1 x 1 µm², 30 nm height scale) with the corresponding line profiles of ALG1 and ALG1-Hept-TertB DS = 0.40 films.

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The CMC and ALG films are superhydrophilic (WCA tends to 0), and the grafting of hydrophobic residues reduces their hydrophilicity by increasing the WCA values. By focusing on CMC-But-TertB and ALG1-But-TertB series, the WCA reveal a close relationship between the DS and the wettability since the increasing of DS leads to a WCA increase. WCA up to 43° is obtained for a DS = 0.44, as a result of the location of the grafted substituents at the interface with air. This trend is noticed for all modified polysaccharides: for a given structure, the increase of the number of grafted substituents yields a WCA increase. Regarding the impact of the structure of the grafts, it appears that for a given DS, the increase of the chain length of the fatty aldehyde and the grafting of bulky norbornene moieties leads to a rise of the water-repellence of the film. Interestingly, the grafting of heptaldehyde and *tert*-butyl isocyanide onto alginate chains at high DS of 0.41 results in a substantial enhancement of the hydrophobicity reflected by a high WCA of 104°. This pronounced gain in hydrophobicity can be not only attributed to the high number of grafted hydrophobic residues per polysaccharide chain as well as their structure (long aliphatic chain), but also to the topologic aspects/roughness of the film surface. Indeed, as shown by AFM image, the film exhibits nanodots uniformly distributed onto the surface with a roughness around 3.5 nm, compared to the film prepared from ALG1 bearing shorter aliphatic chains which displays a featureless surface morphology at the observed scale. The Passerini modification of the polymer allows to provide two hydrophobic
substituents per chemical ligation which are prone to be involved into interchain hydrophobic interactions, at the origin of the presence of associated/aggregated domains displayed by AFM. The wettability is mainly dictated by the two grafted hydrophobic substituents present at the extreme surface (first few angstroms), which confers an “umbrella” effect to the films. By considering the hydrolysis study described in Figure 6, these films should be preferentially used for applications under air at 25°C (as packaging for instance) in order to minimize hydrolysis.

In order to gain insight into the film formation mechanism, QCM-D analysis were undertaken. A gold sensor was firstly covered by a PAH layer (in NaCl at 0.1 M). This strongly physisorbed PAH layer is known to constitute a hydrophilic and positively charged layer enabling to favor the adsorption of polyanions, such as CMC and ALG. After a rinsing step, solutions of CMC, ALG1 and Passerini modified derivatives were injected until the equilibrium of deposition was reached, and a rinsing step with NaCl solution was then applied. The use of salt allows for a partial screening of the electrostatic repulsive interactions which promotes the adsorption at the liquid/liquid interface (Moreau et al. 2012). The evolution of $\Delta f_n/n$ and $\Delta D_n$ (n = 5) was then monitored versus time, for CMC and ALG1 and for the derivatives functionalized with -But and -TertB substituents, at various DS (DS = 0.19, 0.35, 0.44 for CMC, DS = 0.21, 0.31, 0.40 for ALG1) (Fig. 8A and 8B). ALG1 was preferred to ALG2 for QCM-D analysis due to its lower viscosity. The “D-f plots” show that the Sauerbrey equation valuable for rather rigid layers can be exploited in our case to quantitatively determine the mass of adsorbed polymer (Fig. S19) (see Table 3) (Reviakine, Johannsmann, and Richter 2011).

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**Fig. 8.** A) Normalized frequency ($\Delta f_n/n$) and dissipation ($\Delta D_n/n$) changes for the overtone n=5 of the gold substrates, exposed to anchoring PAH and to CMC (in black) and CMC-But-TertB with various DS (DS=0.19 in yellow, DS=0.35 in green and DS=0.44 in blue), B) Normalized frequency ($\Delta f_n/n$) and dissipation ($\Delta D_n/n$) changes for the overtone n=5 of the gold substrates, exposed to anchoring PAH and ALG1 (in black) and ALG1-But-TertB with various DS (DS=0.33 in yellow, DS=0.40 in green).</figcaption></figure>

<table><caption>**Table 3.** Characteristics of CMC and ALG1 layers obtained from QCM-D experiments, after rinsing.</caption><thead><tr><th>entry</th><th>sample</th><th>C (g/L)</th><th>Δf<sub>n</sub>/n (Hz)</th><th>ΔD<sub>n</sub>/n (x 10<sup>-6</sup>)</th><th>Δm (mg/m<sup>2</sup>)</th></tr></thead><tbody><tr><td>1</td><td>CMC</td><td>1.5</td><td>-31.26 ± 0.02</td><td>4.80 ± 0.01</td><td>5.59 ± 0.3</td></tr><tr><td>2</td><td>CMC-But-TertB<sub>0.19</sub></td><td>1.5</td><td>-39.97 ± 0.02</td><td>5.10 ± 0.01</td><td>7.15 ± 0.3</td></tr></tbody></table>
<table><tr><td>3</td><td>CMC-But-TertB<sub>0.35</sub></td><td>1.5</td><td>-48.10 ± 0.02</td><td>6.00 ± 0.01</td><td>8.60 ± 0.3</td></tr><tr><td>4</td><td>CMC-But-TertB<sub>0.44</sub></td><td>1.5</td><td>-49.72 ± 0.02</td><td>7.83 ± 0.01</td><td>8.90 ± 0.3</td></tr><tr><td>5</td><td>CMC-Norb-TertB<sub>0.24</sub></td><td>0.5</td><td>-45.10 ± 0.03</td><td>4.93 ± 0.01</td><td>8.09 ± 0.5</td></tr><tr><td>6</td><td>CMC-Hept-TertB<sub>0.19</sub></td><td>0.5</td><td>-36.40 ± 0.03</td><td>4.40 ± 0.01</td><td>6.46 ± 0.3</td></tr><tr><td>7</td><td>ALG1</td><td>1.5</td><td>-23.61 ± 0.02</td><td>2.35 ± 0.01</td><td>4.22 ± 0.3</td></tr><tr><td>8</td><td>ALG1-But-TertB<sub>0.31</sub></td><td>1.5</td><td>-43.46 ± 0.02</td><td>4.53 ± 0.01</td><td>7.77 ± 0.3</td></tr><tr><td>9</td><td>ALG1-But-TertB<sub>0.40</sub></td><td>1.5</td><td>-45.16 ± 0.02</td><td>5.23 ± 0.01</td><td>8.62 ± 0.4</td></tr></table>

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The first decrease in $\Delta f_n/n$ indicates a rapid deposition of PAH, and this layer is not desorbed through the rinsing step. The second sharp drop of $\Delta f_n/n$, induced after each polymer injection, demonstrates the efficient and very fast adsorption of the polysaccharides onto the surface of PAH. For all samples, an equilibrium of adsorption is reached after 40 and 30 min for CMC and ALG1 derivatives, respectively. Interestingly, in the case of the modified polysaccharides, no noticeable shift of the signals is observed during the rinsing step, which indicates that the polymer adsorption is irreversible in the time scale of the experiments, and that strong interactions are formed at the interface (mainly electrostatic interactions). In particular, $\Delta f_n/n$ values (Table 3, Entries 1-4) vary from -31 Hz for CMC, to -39.9 Hz, - 48.1 Hz and - 49.7 Hz for CMC-But-TertB of DS = 0.19, 0.35 and 0.44, respectively. Regarding the ALG1 series, $\Delta f_n/n$ values (Table 3, Entries 7-9) decrease to -23.6 Hz for unmodified alginate to - 43.4 Hz and - 45.1 Hz for ALG1-But-TertB of DS = 0.31 and 0.40, respectively. The observed difference in frequency shifts underpins the effect of the polysaccharide and the influence of the covalent grafting on the adsorption. The mass of adsorbed polymer was estimated from the Sauerbrey equation (Experimental Section) (Table 3). Firstly, we can see that the Passerini derivatization does not alter the adsorption ability of the polysaccharides, but do the opposite the adsorption is significantly enhanced by the chemical functionalization. The adsorbed mass is increased from 559 ng/cm$^2$ up to 715 ng/cm$^2$ for CMC-But-TertB of DS = 0.19, and the mass increasing is all the more pronounced that the DS is high. This beneficial effect of the chemical modification on the adsorption is also observed for the alginate-based derivatives, and the mass increases from 422 ng/cm$^2$ for ALG1 up to 862 ng/cm$^2$ for ALG1-But-TertB of DS = 0.40 (Table 3, entries 7-9). The enhancement of the adsorbed masses is accompanied by a slight increase of the dissipation of energy (Table 3, $\Delta D_n$ from $\approx 4.8 \times 10^{-6}$ for CMC up to $\approx 7.83 \times 10^{-6}$ for CMC-But-TertB of DS = 0.44, and from $2.35 \times 10^{-6}$ for ALG1 up to $5.23 \times 10^{-6}$ for ALG1-But-TertB of DS = 0.40). The increase of the adsorbed mass with the chemical functionalization may appear counter-intuitive since the modified polysaccharides have a lower number of negatively charged carboxylate units due to the substitution by hydrophobic substituents, which would be detrimental for the development of electrostatic interactions with the PAH sub-layer. The Passerini modification of CMC and ALG by aliphatic substituents naturally leads to an increase of the average molar mass of the modified repetitive units, which could have explained the enhanced deposited mass observed for the chemically modified products. Thus, we calculated $\Delta n$ values, obtained from the ratio between $\Delta m$ and the molar mass (table, Fig. S20), and the results reveal that similar increase in the deposited polymer is observed even after the “normalization” by the molar mass of the repetitive unit. Thus, the increase of the molar mass of the modified sugar unit alone cannot explain the increase of the
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amount of deposited polymer. Consequently, it is reasonable to assume that the larger amount of adsorbed polymer arises from a change of the chain conformation adopted by the functionalized CMC and ALG1 in water and the chain conformation is all the more modified than the chemical functionalization is high. These results are in concordance with our previous on the CMC deposition onto amorphous cellulose surfaces (Pettignano, Daunay, et al. 2019). The presence of twofold hydrophobic substituents might result in the formation of interchain hydrophobic interactions. This leads to more tightly coiled chains in solution, which are deposited in a scattered manner, thus inducing a favorable packing of the adsorbed chains onto the surface. This hypothesis is confirmed by AFM analysis (see below, Fig. 9 and 10). The slight increase of energy dissipation observed for polysaccharides modified by But-TertB moieties might be counterintuitive. In fact, a balance between competitive phenomena must be considered: i) the increasing of hydrophobicity degree and the reducing of the number of carboxylate groups are expected to lower the water sorption that might contribute to decrease $\Delta D_n$, while ii) the presence of polymer loops/tails and the formation of aggregated zones within the deposited layer can favor the capture of additional water molecules that should contribute to increase $\Delta D_n$ (Zhang 2014). It is worth to mention that the deposition of ALG and its derivatives leads to lower deposited amounts, compared to the CMC series, due to the lower molecular weight of ALG1.

The QCM-D experiments were extended to more hydrophobic CMC-based dually functionalized: CMC-Norb-TertB (DS=0.24) and CMC-Hept-TertB (DS=0.19) (entries 6 and 7, Table 3, Fig. S21) and provided slightly lower $\Delta f_n/n$ values, and especially weaker $\Delta D_n$ values, that becomes even lower than for CMC. In this case, the hydrophobicity afforded by the grafting of longer aliphatic chain (in C7) predominates and reduces the water uptake.

In order to examine the surface morphology and to gain insight into the deposition mode of the polysaccharides, the coated QCM-D sensors, submitted to a rinsing step to remove residual salt and to a drying step, were analyzed by AFM in Tapping mode. The images, at dried state, were not conclusive and no discrimination about surface morphologies could be observed between the native sensors and the sensors covered by polysaccharides. Indeed, the very low thickness (≈10 nm) of the deposited layer as well as the inherent roughness ($Rq$ = 1 nm) of the bare gold surface did not allow to evidence noticeable changes of topography. In order to gain in sensitivity, AFM in PeakForce Tapping mode images, not only acquired through a controlled apply force but also under a localized water submersion, were collected. Thus, differences at swollen state can be visualized (Figures 9 and 10).
<figure><img src="image_20.png" /><figcaption>**Fig. 9.** AFM height images (PeakForce Tapping mode) upon water medium of a bare gold sensor (A) and gold sensors exposed to QCM-D experiments to the injection of PAH, followed by injection of CMC sodium salt (B), CMC-But-*Tert*B DS=0.44 (C), or CMC-Hept-*Tert*B DS = 0.13 (D).</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>**Fig. 10.** AFM height images (PeakForce Tapping mode) upon water medium of a gold sensor exposed to QCM-D experiments to the injection of PAH, followed by injection of CMC-Hept-*Tert*B DS = 0.13.</figcaption></figure>

The gold sensors exhibit a roughness around 1 nm (Figure 9A). The gold nanoparticles are still visible after the successive depositions of PAH and CMC, but the presence of polymers can be identified upon the form of dispersed very small nanodots (Figure 9 B). After the PAH and CMC-But-*Tert*B (DS = 0.44) deposition, larger aggregates (up to 130 nm in width) are observed, yielding a roughness of 1.85 nm (Figure 9C). These
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nanoprotrusions seem to have the same adhesive and nano-mechanical properties than the rest of the surface sample. Regarding the deposition of CMC-Hept-TertB (DS = 0.13) some small surfacic dots (width around nm) also emerge (Figures 9C and 10). The presence of such aggregates observed upon humid state after the deposition of the modified polysaccharides arises from the formation of hydrophobic interactions in water between the grafted moieties, i.e. butyl and tert-butyl groups. This validates our previous hypothesis concerning the deposition mode: the functionalized polysaccharides form more tightly coiled chains in solution that conducts to a more compact deposition and to a significant increase of the deposited polymer amount, as demonstrated by the mass values gathered in Table 3.

## 4. CONCLUSION

P 3-CR was successfully applied to carboxymethyl cellulose and alginates in aqueous conditions, to lead to never described twofold hydrophobized polysaccharides derivatives bearing aliphatic ramified or bulky moieties, or fatty chains with various length. DS up to around 0.50 were obtained, that corresponds to a substantial conversion (≈ 70%) of carboxylic groups. The Passerini reactivity is enhanced by an increase of the amount of reactants and is altered for long aliphatic chain reactants due to water solubility issues. As hypothesized in the introduction, the Passerini hydrophobized polysaccharides were successfully processed as thin films exhibiting tuneable wettability properties and pronounced hydrophobicity (WCA > 90°) was obtained, from the result of both hydrophobicity and adapted roughness related to the formation of aggregated zones. Interestingly, from QCM-D experiments, it appeared that the grafting of aliphatic substituents significantly increases the mass of adsorbed polymer, due to the formation of hydrophobic interactions that induce a favourable packing of the deposited chains, as confirmed by AFM PeakForce Tapping analysis. Also, the degrees of hydration of the films are controlled by the balance between the hydrophobicity degree and the conformations of the chains within the film. Such twofold hydrophobized derivatives could find applications in various domains such as biomedical or cosmetic fields (bioadhesives, formulations for drug delivery, surfactants, compatibilizing reagents, interfacial agents) or packaging field (polymer films used as coatings).
644 ASSOCIATED CONTENT

645 **Declaration of Competing Interest**

646 The authors declare no competing financial interest.

647 **Funding Sources**

648 This work was supported by the Agence Nationale de la Recherche (ANR) in France as part of the project

649 MODERATO (Grant number: ANR-21-CE06-0035).

650 **Acknowledgments**

651 The authors thank the Agence Nationale de la Recherche (ANR) in France for the financial support of this

652 project MODERATO (Grant number: ANR-21-CE06-0035). The authors acknowledge the Consortium Lyon

653 Saint-Etienne de Microscopie (CLYM, FED 4092) for the access to the microscopes. They are grateful for

654 Fernande Da Cruz-Boisson, Carlos Fernandes de Alba and Patrick Goetinck for precious advices in NMR

655 experiments, and Laurent Remy for fruitful discussions on Passerini reactions.

656 **References**

657 Abu-Rabeah, Khalil, Boris Polyak, Rodica E. Ionescu, Serge Cosnier, and Robert S. Marks. 2005.

658 “Synthesis and Characterization of a Pyrrole–Alginate Conjugate and Its Application in a

659 Biosensor Construction.” *Biomacromolecules* 6(6):3313–18. doi: 10.1021/bm050339j.

660 Andresen, Inger-Lill, Terence Painter, and Olav Smidsrød. 1977. “Concerning the Effect of

661 Periodate Oxidation upon the Intrinsic Viscosity of Alginate.” *Carbohydrate Research*

662 59(2):563–66. doi: 10.1016/S0008-6215(00)83194-8.

663 Batelaan, Jan Gerardus, and Peter Marten Van Der Horst. 2000. “Method of Making Amide

664 Modified Carboxyl-Containing Polysaccharide and Fatty Amide-Modified Polysaccharide

665 so Obtainable.”

666 Benalaya, Ikbel, Gilberto Alves, João Lopes, and Luís R. Silva. 2024. “A Review of Natural

667 Polysaccharides: Sources, Characteristics, Properties, Food, and Pharmaceutical

668 Applications.” *International Journal of Molecular Sciences* 25(2):1322. doi:

669 10.3390/ijms25021322.

670 Bertagnolli, Caroline, Ana Paula D. M. Espindola, Sirlei Jaiana Kleinübing, Ljubica Tasic, and

671 Meuris Gurgel Carlos da Silva. 2014. “Sargassum Filipendula Alginate from Brazil:

672 Seasonal Influence and Characteristics.” *Carbohydrate Polymers* 111:619–23. doi:

673 10.1016/j.carbpol.2014.05.024.

674 Bordenave, Nicolas, Stephane Grelier, and Veronique Coma. 2010. “Hydrophobization and

675 Antimicrobial Activity of Chitosan and Paper-Based Packaging Material.”

676 *Biomacromolecules* 11(1):88–96. doi: 10.1021/bm9009528.

677 Broderick, Eilish, Henry Lyons, Tony Pembroke, Hugh Byrne, Brian Murray, and Michael Hall.

678 2006. “The Characterisation of a Novel, Covalently Modified, Amphiphilic Alginate

679 Derivative, Which Retains Gelling and Non-Toxic Properties.” *Journal of Colloid and

680 *Interface Science* 298(1):154–61. doi: 10.1016/j.jcis.2005.12.026.
681 Bu, Huaitian, Anna-Lena Kjøniksen, Kenneth D. Knudsen, and Bo Nyström. 2004. "Rheological and Structural Properties of Aqueous Alginate during Gelation via the Ugi Multicomponent Condensation Reaction." *Biomacromolecules* 5(4):1470-79. doi: 10.1021/bm049947+.

684 Carré, Marie-Christiane, Christine Delestre, Patrick Hubert, and Edith Dellacherie. 1991. "Covalent Coupling of a Short Polyether on Sodium Alginate: Synthesis and Characterization of the Resulting Amphiphilic Derivative." *Carbohydrate Polymers* 16(4):367-79. doi: 10.1016/0144-8617(91)90055-H.

688 Chamberlain, N. H., G. E. Cunningham, and J. B. Speakman. 1946. "Alginic Acid Diacetate." *Nature* 158(4016):553-553. doi: 10.1038/158553b0.

690 Charpentier, D., G. Mocanu, A. Carpov, S. Chapelle, L. Merle, and G. Müller. 1997. "New Hydrophobically Modified Carboxymethylcellulose Derivatives." *Carbohydrate Polymers* 33(2):177-86. doi: 10.1016/S0144-8617(97)00031-3.

693 Chen, Xiuqiong, Qingmei Zhu, Chang Liu, Dongze Li, Huiqiong Yan, and Qiang Lin. 2021. "Esterification of Alginate with Alkyl Bromides of Different Carbon Chain Lengths via the Bimolecular Nucleophilic Substitution Reaction: Synthesis, Characterization, and Controlled Release Performance." *Polymers* 13(19):3351. doi: 10.3390/polym13193351.

697 Coleman, Robert J., Gwen Lawrie, Lynette K. Lambert, Michael Whittaker, Kevin S. Jack, and Lisbeth Grøndahl. 2011. "Phosphorylation of Alginate: Synthesis, Characterization, and Evaluation of in Vitro Mineralization Capacity." *Biomacromolecules* 12(4):889-97. doi: 10.1021/bm1011773.

701 Čopíková, Jana, Tomáš Taubner, Jan Tůma, Andriy Synytsya, Dagmar Dušková, and Milan Marounek. 2015. "Cholesterol and Fat Lowering with Hydrophobic Polysaccharide Derivatives." *Carbohydrate Polymers* 116:207-14. doi: 10.1016/j.carbpol.2014.05.009.

704 Deng, Yaling, Amin Shavandi, Oseweuba Valentine Okoro, and Lei Nie. 2021. "Alginate Modification via Click Chemistry for Biomedical Applications." *Carbohydrate Polymers* 270:118360. doi: 10.1016/j.carbpol.2021.118360.

707 Esen, Eren, and Michael A. R. Meier. 2020. "Sustainable Functionalization of 2,3-Dialdehyde Cellulose via the Passerini Three-Component Reaction." *ACS Sustainable Chemistry & Engineering*. doi: 10.1021/acssuschemeng.0c06153.

710 Gabriel, Lars, and Thomas Heinze. 2018. "Diversity of Polysaccharide Structures Designed by Aqueous Ugi-Multi-Compound Reaction." *Cellulose* 25(5):2849-59. doi: 10.1007/s10570-018-1754-y.

713 Galant, Céline, Anna-Lena Kjøniksen, Giao T. M. Nguyen, Kenneth D. Knudsen, and Bo Nyström. 2006. "Altering Associations in Aqueous Solutions of a Hydrophobically Modified Alginate in the Presence of β-Cyclodextrin Monomers." *The Journal of Physical Chemistry B* 110(1):190-95. doi: 10.1021/jp0518759.

717 Gomez, Cesar G., Gérard Chambat, Alain Heyraud, Marcelo Villar, and Rachel Auzély-Velty. 2006. "Synthesis and Characterization of a β-CD-Alginate Conjugate." *Polymer* 47(26):8509-16. doi: 10.1016/j.polymer.2006.10.011.

720 Grasdalen, Hans, Bjørn Larsen, and Olav Smidsrød. 1979. "A p.m.r. Study of the Composition and Sequence of Uronate Residues in Alginates." *Carbohydrate Research* 68(1):23-31. doi: 10.1016/S0008-6215(00)84051-3.
723 Hamzah, Oussama, Tom Vandenbrouck, Laurent Heux, and Bruno Jean. 2024. "Insight into the
724 Hydrophobic Functionalization of Cellulose Microfibrils Using the Passerini Three-
725 Component Reaction." *Carbohydrate Polymers* 341:122323. doi:
726 10.1016/j.carbpol.2024.122323.

727 Hartman, Jonas, Ann-Christine Albertsson, and John Sjöberg. 2006. "Surface- and Bulk-Modified
728 Galactoglucomannan Hemicellulose Films and Film Laminates for Versatile Oxygen
729 Barriers." *Biomacromolecules* 7(6):1983-89. doi: 10.1021/bm060129m.

730 Heinze, T., and T. Liebert. 2001. "Unconventional Methods in Cellulose Functionalization."
731 *Progress in Polymer Science* 26(9):1689-1762. doi: 10.1016/S0079-6700(01)00022-3.

732 Kang, Hyun-Ah, Gyu-Jong Jeon, Moo-Yeal Lee, and Ji-Won Yang. 2002. "Effectiveness Test of
733 Alginate-Derived Polymeric Surfactants." *Journal of Chemical Technology &
734 Biotechnology* 77(2):205-10. doi: 10.1002/jctb.550.

735 Kang, Hyun-Ah, Moon Sik Shin, and Ji-Won Yang. 2002. "Preparation and Characterization of
736 Hydrophobically Modified Alginate." *Polymer Bulletin* 47(5):429-35. doi:
737 10.1007/s00289020005.

738 Khine, Yee Yee, Sylvia Ganda, and Martina H. Stenzel. 2018. "Covalent Tethering of Temperature
739 Responsive pNIPAm onto TEMPO-Oxidized Cellulose Nanofibrils via Three-Component
740 Passerini Reaction." *ACS Macro Letters* 7(4):412-18. doi: 10.1021/acsmacrolett.8b00051.

741 Kim, Han-Sem, Minsoo Song, Eun-Jung Lee, and Ueon Sang Shin. 2015. "Injectable Hydrogels
742 Derived from Phosphorylated Alginic Acid Calcium Complexes." *Materials Science and
743 Engineering: C* 51:139-47. doi: 10.1016/j.msec.2015.02.031.

744 Leonard, M., M. Rastello De Boisseson, P. Hubert, F. Dalençon, and E. Dellacherie. 2004.
745 "Hydrophobically Modified Alginate Hydrogels as Protein Carriers with Specific Controlled
746 Release Properties." *Journal of Controlled Release* 98(3):395-405. doi:
747 10.1016/j.jconrel.2004.05.009.

748 Li, Yimeng, Małgorzata Milewska, Yee Yee Khine, Nicholas Ariotti, and Martina H. Stenzel. 2022.
749 "Trehalose Coated Nanocellulose to Inhibit the Infections by S. Aureus." *Polymer Chemistry*
750 13(11):1502-9. doi: 10.1039/D1PY01422F.

751 Lin, Chien-Chi, Chang Seok Ki, and Han Shih. 2015. "Thiol-Norbornene Photoclick Hydrogels for
752 Tissue Engineering Applications." *Journal of Applied Polymer Science* 132(8):app.41563.
753 doi: 10.1002/app.41563.

754 Moreau, C., N. Beury, N. Delorme, and B. Cathala. 2012. "Tuning the Architecture of Cellulose
755 Nanocrystal-Poly(Allylamine Hydrochloride) Multilayered Thin Films: Influence of
756 Dipping Parameters." *Langmuir* 28(28):10425-36. doi: 10.1021/la301293r.

757 Nooy, Arjan de. 1999. "Versatile Synthesis of Polysaccharide Hydrogels Using the Passerini and
758 Ugi Multicomponent Condensations." *Macromolecules*.

759 Onwukamike, Kelechukwu N., Stéphane Grelier, Etienne Grau, Henri Cramail, and Michael A. R.
760 Meier. 2019. "Critical Review on Sustainable Homogeneous Cellulose Modification: Why
761 Renewability Is Not Enough." *ACS Sustainable Chemistry & Engineering* 7(2):1826-40.
762 doi: 10.1021/acssuschemeng.8b04990.

763 Pawar, Siddhesh N., and Kevin J. Edgar. 2011. "Chemical Modification of Alginates in Organic
764 Solvent Systems." *Biomacromolecules* 12(11):4095-4103. doi: 10.1021/bm201152a.
765 Pawar, Siddhesh N., and Kevin J. Edgar. 2013. "Alginate Esters via Chemoselective Carboxyl Group Modification." *Carbohydrate Polymers* 98(2):1288-96. doi: 10.1016/j.carbpol.2013.08.014.

768 Pelletier, S., P. Hubert, E. Payan, P. Marchal, L. Choplin, and E. Dellacherie. 2001. "Amphiphilic Derivatives of Sodium Alginate and Hyaluronate for Cartilage Repair: Rheological Properties." *Journal of Biomedical Materials Research* 54(1):102-8. doi: 10.1002/1097-4636(200101)54:1<102::aid-jbm12>3.0.co;2-1.

772 Pettignano, Asja, Daniel Antonio Aguilera, Nathalie Tanchoux, Luca Bernardi, and Françoise Quignard. 2019. "Chapter 17 - Alginate: A Versatile Biopolymer for Functional Advanced Materials for Catalysis." Pp. 357-75 in *Studies in Surface Science and Catalysis*. Vol. 178, *Horizons in Sustainable Industrial Chemistry and Catalysis*, edited by S. Albonetti, S. Perathoner, and E. A. Quadrelli. Elsevier.

777 Pettignano, Asja, Aurélia Charlot, and Etienne Fleury. 2019. "Carboxyl-Functionalized Derivatives of Carboxymethyl Cellulose: Towards Advanced Biomedical Applications." *Polymer Reviews* 59(3):510-60. doi: 10.1080/15583724.2019.1579226.

780 Pettignano, Asja, Adélie Daunay, Céline Moreau, Bernard Cathala, Aurélia Charlot, and Etienne Fleury. 2019. "Sustainable Modification of Carboxymethyl Cellulose by Passerini Three-Component Reaction and Subsequent Adsorption onto Cellulosic Substrates." *ACS Sustainable Chemistry & Engineering* 7(17):14685-96. doi: 10.1021/acssuschemeng.9b02634.

785 Pettignano, Asja, Julien Leguy, Laurent Heux, Bruno Jean, Aurélia Charlot, and Etienne Fleury. 2020. "Multifunctionalization of Cellulose Microfibrils through a Cascade Pathway Entailing the Sustainable Passerini Multi-Component Reaction." *Green Chemistry* 22(20):7059-69. doi: 10.1039/D0GC02532A.

789 Polyak, Boris, Shimona Geresh, and Robert S. Marks. 2004. "Synthesis and Characterization of a Biotin-Alginate Conjugate and Its Application in a Biosensor Construction." *Biomacromolecules* 5(2):389-96. doi: 10.1021/bm034454a.

792 Rastello De Boisseson, M., M. Leonard, P. Hubert, P. Marchal, A. Stequert, C. Castel, E. Favre, and E. Dellacherie. 2004. "Physical Alginate Hydrogels Based on Hydrophobic or Dual Hydrophobic/Ionic Interactions: Bead Formation, Structure, and Stability." *Journal of Colloid and Interface Science* 273(1):131-39. doi: 10.1016/j.jcis.2003.12.064.

796 Remy, Laurent, Guillaume Sudre, Aurélia Charlot, and Etienne Fleury. 2023. "α-Substituted Ketones as Reagent for Passerini Modification of Carboxymethyl Cellulose: Toward Dually Functionalized Derivatives and Thermo-Sensitive Chemical Hydrogels." *Carbohydrate Polymers* 320:121228. doi: 10.1016/j.carbpol.2023.121228.

800 Reviakine, Ilya, Diethelm Johannsmann, and Ralf P. Richter. 2011. "Hearing What You Cannot See and Visualizing What You Hear: Interpreting Quartz Crystal Microbalance Data from Solvated Interfaces." *Analytical Chemistry* 83(23):8838-48. doi: 10.1021/ac201778h.

803 Roschangar, F., R. A. Sheldon, and C. H. Senanayake. 2015. "Overcoming Barriers to Green Chemistry in the Pharmaceutical Industry – the Green Aspiration Level™ Concept." *Green Chemistry* 17(2):752-68. doi: 10.1039/C4GC01563K.

806 Sauerbrey, G. 1991. *The Use of Quartz Crystal Oscillators for Weighing Thin Layers and for Microweighing Applications*.
808 Sinquin, A., M. C. Houzelle, P. Hubert, L. Choplin, M. L. Viriot, and E. Dellacherie. 1996. "Amphiphilic Derivatives of Propylene Glycol Alginate: A Revisit of Their Physicochemical Behavior in Dilute Aqueous Solution." *Langmuir* 12(16):3779-82. doi: 10.1021/la951036l.

812 Sinquin, A., P. Hubert, P. Marchal, L. Choplin, and E. Dellacherie. 1996. "Rheological Properties of Semi-Dilute Aqueous Solutions of Hydrophobically Modified Propylene Glycol Alginate Derivatives." *Colloids and Surfaces A: Physicochemical and Engineering Aspects* 112(2):193-200. doi: 10.1016/0927-7757(96)03513-3.

816 SkjÅk-Bræk, Gudmund, Sergio Paoletti, and Teresa Gianferrara. 1989. "Selective Acetylation of Mannuronic Acid Residues in Calcium Alginate Gels." *Carbohydrate Research* 185(1):119-29. doi: 10.1016/0008-6215(89)84027-3.

819 Skjåk-Bræk, Gudmund, Flavio Zanetti, and Sergio Paoletti. 1989. "Effect of Acetylation on Some Solution and Gelling Properties of Alginates." *Carbohydrate Research* 185(1):131-38. doi: 10.1016/0008-6215(89)84028-5.

822 Söyler, Z., K. N. Onwukamike, S. Grelier, E. Grau, H. Cramail, and M. A. R. Meier. 2018. "Sustainable Succinylation of Cellulose in a CO2-Based Switchable Solvent and Subsequent Passerini 3-CR and Ugi 4-CR Modification." *Green Chemistry* 20(1):214-24. doi: 10.1039/C7GC02577G.

826 Thabet, Omar A., Faisal S. Al Muzini, Abdulaziz M. Atiya, Khalid A. Alamry, Mahmoud A. Hussein, and Richard Hoogenboom. 2023. "Hydrophobic Carboxymethyl Cellulose as a Clean-up Sorbent in the Determination of Nitrofuran Metabolites in Animal-Fat Samples." *RSC Advances* 13(47):33221-30. doi: 10.1039/D3RA07021B.

830 Tizzotti, Morgan, Aurelia Charlot, Etienne Fleury, Martina Stenzel, and Julien Bernard. 2010. "Modification of Polysaccharides through Controlled/Living Radical Polymerization Grafting-towards the Generation of High Performance Hybrids." *Macromolecular Rapid Communications* 31(20):1751-72. doi: 10.1002/marc.201000072.

834 Vallée, Frédéric, Christophe Müller, Alain Durand, Sarah Schimchowitsch, Edith Dellacherie, Christian Kelche, Jean Christophe Cassel, and Michèle Leonard. 2009. "Synthesis and Rheological Properties of Hydrogels Based on Amphiphilic Alginate-Amide Derivatives." *Carbohydrate Research* 344(2):223-28. doi: 10.1016/j.carres.2008.10.029.

838 Vleugels, Leo F. W., Stella Ricois, Ilja K. Voets, and Remco Tuinier. 2018. "Determination of the 'Apparent pKa' of Selected Food Hydrocolloids Using Ortho-Toluidine Blue." *Food Hydrocolloids* 81:273-83. doi: 10.1016/j.foodhyd.2018.02.049.

841 Wang, Wei, Chengrong Qin, Wei Li, Jiayan Ge, and Chengqi Feng. 2020. "Improving Moisture Barrier Properties of Paper Sheets by Cellulose Stearoyl Ester-Based Coatings." *Carbohydrate Polymers* 235:115924. doi: 10.1016/j.carbpol.2020.115924.

844 Wassermann, Albert. 1946. "Alginic Acid-Acetate." *Nature* 158(4008):271-271. doi: 10.1038/158271a0.

846 Yang, Ji Sheng, Qi Quan Zhou, and Wen He. 2013. "Amphipathicity and Self-Assembly Behavior of Amphiphilic Alginate Esters." *Carbohydrate Polymers* 92(1):223-27. doi: 10.1016/j.carbpol.2012.08.100.

849 Yang, Jun, Mitsuaki Goto, Hirohiko Ise, Chong-Su Cho, and Toshihiro Akaike. 2002. "Galactosylated Alginate as a Scaffold for Hepatocytes Entrapment." *Biomaterials* 23(2):471-79. doi: 10.1016/s0142-9612(01)00129-6.
852 Zabivalova, N., A. Bochek, L. Kalyuzhnaya, E. Vlasova, and B. Volchek. 2003. "Carboxymethyl
853 Cellulose Amides and Their Properties." *Russian Journal of Applied Chemistry - RUSS J
854 APPL CHEM-ENG TR* 76:1998–2002. doi: 10.1023/B:RJAC.0000022456.23088.b6.

855 Zhang, Xiao. 2014. "Adsorption of Biomacromolecules onto Polysaccharide Surfaces." Virginia
856 Polytechnic Institute and State University.

857 Zhu, Huiguang, Jian Ji, Rongyi Lin, Changyou Gao, Linxian Feng, and Jiacong Shen. 2002.
858 "Surface Engineering of Poly(Dl-Lactic Acid) by Entrapment of Alginate-Amino Acid
859 Derivatives for Promotion of Chondrogenesis." *Biomaterials* 23(15):3141–48. doi:
860 10.1016/S0142-9612(02)00058-3.

861

862 SUPPORTING INFORMATION

863

864

<figure><img src="image_22.png" /></figure>

865

866

867 **Fig. S1.** Conductometric titration of CMC and ALG1 at 1.5 g/L in water by using a HCl solution at 0.33M.

868

869 *Calculation of the DS in COONa groups.*

870 Three domains of evolution are observed in Fig. S1 and correspond to: (I) the reaction with free NaOH
871 (strong base), (II) the protonation of the carboxylate functions of the polysaccharide (weak base) and (III)
872 the presence in excess of HCl. The extrapolation of these domains gives two intersections at V₁ and V₂ of
873 HCl. The use of Eq. 1 and 2 allows to access DS through Eq. 1-2.

874

875

$$
A = \frac{(V_2 - V_1) \times 0.33}{m} \qquad (1)
$$
$$DS = \frac{A \times M_{UNIT}}{(1 - \Delta M_{COONa}A)} \quad (2)$$

Where m refers to the mass of dry polysaccharide, MUNIT to the molar mass of a sugar unit (0.162 kg/mol for CMC and 0.148 kg/mol for ALG), and ΔMCOONa to the gain in molar mass corresponding to the substitution of an -OH group by a carboxylate or a carboxymethylate group (0.080 kg/mol).

The titrations allowed to calculate the number of initial carboxylate groups, and values of 1.25 for CMC and 0.95 for ALG were found that are very close to the values provided by the supplier.

<figure><img src="image_23.png" /><figcaption>Fig. S2. SEC curves of A) CMC B) ALG1 before (Na-in red) and after (H- in black) acidification and purification steps</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>Fig. S3. HSQC NMR spectra of sample ALG1-But-TertB (Table 2, Entry 2), recorded in D2O/Acetonitrile 50/50 v/v% at 323K.</figcaption></figure>
<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /><figcaption>**Fig. S4.** A) ATR-IR of *i*) CMC, *ii*) partially protonated CMC, *iii*) CMC-But-CH (Table 1, Entry 4) and B) ¹H NMR (512 scans, D1=10s) recorded in D₂O at 298K.</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>**Fig. S5.** A) ATR-IR of *i*) CMC, *ii*) partially protonated CMC, *iii*) CMC-But-TertB (Table 1, Entry 1) and B) ¹H NMR (512 scans, D1=10s) recorded in D₂O at 298K.</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>**Fig. S6.** A) ATR-IR of *i*) CMC, *ii*) partially protonated CMC, *iii*) CMC-Hept-TertB (Table 1, Entry 8) and
B) ¹H NMR (512 scans, D1=10s) recorded in D₂O/DMSO-d6 (50/50 v/v%) at 298K.</figcaption></figure>
<figure><img src="image_29.png" /><figcaption>**Fig. S7.** A) ATR-IR of i) CMC, ii) partially protonated CMC, iii) CMC-Undeca-TertB (Table 1, Entry 14) and B) ¹H NMR (512 scans, D1=10s) recorded in D₂O/Acetonitrile-d3 (50/50 v/v%) at 298K.</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>**Fig. S8.** A) ATR-IR of i) CMC, ii) partially protonated CMC, iii) CMC-Undece-TertB (Table 1, Entry 16) and B) ¹H NMR (512 scans, D1=10s) recorded in D₂O/Acetonitrile-d3 (50/50 v/v%) at 298K.</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>**Fig. S9.** A) ATR-IR of i) CMC, ii) partially protonated CMC, iii) CMC-Norb-TertB (Table 1, Entry 21) and B) ¹H NMR (512 scans, D₁=10s) recorded in D₂O at 298K.</figcaption></figure>
<figure><img src="image_32.png" /><figcaption>**Fig. S10.** A) ATR-IR of i) ALG1, ii) partially protonated ALG1, iii) ALG1-Hept-TertB (Table 2, Entry 5) and B) ¹H NMR (512 scans, D₁=10s) recorded in D₂O at 298K.</figcaption></figure>
<figure><img src="image_33.png" /><figcaption>**Fig. S11.** A) ATR-IR of i) ALG1, ii) partially protonated ALG1, iii) ALG1-Norb-TertB (Table 2, Entry 9) and B) ¹H NMR (512 scans, D1=10s) recorded in D₂O/Acetonitrile-d3 (50/50 v/v%) at 298K.</figcaption></figure>
<figure><img src="image_34.png" /><figcaption>**Fig. S12.** A) ATR-IR and B) ¹H NMR (512 scans, D1=10s) of ALG2-But-TertB (Table 2, Entry 11), recorded in D₂O/Acetonitrile-d3 (50/50 v/v%) at 333K</figcaption></figure>
<figure><img src="image_35.png" /><figcaption>**Fig. S13.** A) ATR-IR and B) ¹H NMR (512 scans, D1=10s) of ALG2-Hept-TertB (Table 2, Entry 14), recorded in D₂O/Acetonitrile-d3 (50/50 v/v%) at 333K.</figcaption></figure>
<figure><img src="image_36.png" /><figcaption>**Fig. S14.** A) ATR-IR and B) ¹H NMR (512 scans, D1=10s) of ALG2-Norb-*TertB* (Table 2, Entry 16), recorded in D₂O/Acetonitrile-d3 (50/50 v/v%) at 323K.</figcaption></figure>
<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /><figcaption>**Fig. S15.** ¹H NMR study of CMC-But-*Tert*B (Table 1, entry 1) through hydrolysis at A) 323K B) 353K in D₂O.</figcaption></figure>

922

923

924
<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>**Fig. S16.** ¹H NMR study of CMC-Norb-*TertB* (Table 1, entry 20) through hydrolysis at A) 323K B) 353K in D₂O/Acetonitrile-d3 (50/50 v/v%).</figcaption></figure>

925

926

927
<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /><figcaption>**Fig. S17.** ¹H NMR study of ALG1-Norb-*TertB* (Table 2, entry 9) through hydrolysis at A) 323K B) 353K in D₂O/Acetonitrile-d3 (50/50 v/v%).</figcaption></figure>

928

929

930

931
<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /><figcaption>**Fig. S18.** ¹H NMR study of ALG2-Norb-TertB (Table 2, entry 16) through hydrolysis at A) 323K B) 353K in D₂O/Acetonitrile-d3 (50/50 v/v%).</figcaption></figure>
<figure><img src="image_48.png" /><figcaption>**Fig. S19.** Evolution of $\Delta D_n/n$ vs $\Delta f_n/n$ for the overtone n = 5, during the adsorption step of A) CMC (in black) and CMC-But-TertB with various DS (DS=0.19 in yellow, DS=0.35 in green and DS=0.44 in blue), B) ALG1 (in black) and ALG1-But-TertB with various DS (DS=0.33 in yellow, DS=0.40 in green). Exposition of Sauerbrey limit as defined as $\Delta D_n=4.10^{-7}\Delta f_n/n$.</figcaption></figure>

<table><caption>**Fig. S20.** Table of characteristics of CMC and ALG1 layers obtained from QCM-D experiments, after rinsing, with the calculation of $\Delta n$, which corresponds to $\Delta m/M$, M being the average molar mass of the modified repetitive unit.</caption><thead><tr><th>entry</th><th>sample</th><th>C (g/L)</th><th>Δn (mmol/m²)</th></tr></thead><tbody><tr><td>1</td><td>CMC sodium salt</td><td>1.5</td><td>0.022 ± 0.001</td></tr><tr><td>2</td><td>CMC-But-TertB<sub>0.19</sub></td><td>1.5</td><td>0.026 ± 0.001</td></tr><tr><td>3</td><td>CMC-But-TertB<sub>0.35</sub></td><td>1.5</td><td>0.029 ± 0.001</td></tr><tr><td>4</td><td>CMC-But-TertB<sub>0.44</sub></td><td>1.5</td><td>0.029 ± 0.001</td></tr><tr><td>5</td><td>CMC-Norb-TertB<sub>0.24</sub></td><td>0.5</td><td>0.028 ± 0.002</td></tr><tr><td>6</td><td>CMC-Hept-TertB<sub>0.19</sub></td><td>0.5</td><td>0.023 ± 0.001</td></tr><tr><td>7</td><td>ALG1</td><td>1.5</td><td>0.021 ± 0.001</td></tr><tr><td>8</td><td>ALG1-But-TertB<sub>0.31</sub></td><td>1.5</td><td>0.033 ± 0.001</td></tr><tr><td>9</td><td>ALG1-But-TertB<sub>0.40</sub></td><td>1.5</td><td>0.034 ± 0.002</td></tr></tbody></table>
<figure><img src="image_49.png" /><figcaption>**Fig. S21.** A) Normalized frequency (Δf<sub>n</sub>/n) (in black) and dissipation (ΔD<sub>n</sub>/n) (in red) changes for the overtone *n*=5 of the gold substrates, exposed to anchoring PAH and CMC. B) Normalized frequency (Δf<sub>n</sub>/n) (in black) and dissipation (ΔD<sub>n</sub>/n) (in red) changes for the overtone *n*=5 of the gold substrates, exposed to anchoring PAH and CMC-But-TertB DS = 0.19 (in plane line), CMC Hept-TertB DS = 0.19 (in dash line), CMC-Norb-TertB DS = 0.24 (in dotted line).</figcaption></figure>