Molecular Design of Thermoresponsive Cellulose: How Graft Architecture Dictates Sol‐Gel Transitions

ABSTRACT

The development of polysaccharide‐peptoid hybrids represents a promising route toward sustainable, stimuli‐responsive biomaterials. However, achieving controlled synthesis and predictable thermal transitions, particularly reversible sol‐gel behavior, remains challenging. Herein, we report a systematic study on the synthesis of thermoresponsive carboxymethyl cellulose (CMC) graft polymers via the Ugi multicomponent polymerization of glycylglycine as a model dipeptide. Two distinct synthetic strategies, stepwise grafting of preformed n‐propylamine‐capped peptoids and one‐pot grafting, are employed to design graft architecture. Comprehensive characterizations reveal that graft length, density, and molecular weight dictate the thermal response. Notably, only graft polymers with short, dense grafts (CNP_{80 (12.0)}) undergo a reversible sol‐gel transition upon heating, while long, sparse grafts (CNP₅, CNP₂₀) lead to particulate aggregation without gelation. In situ variable‐temperature FTIR, coupled with two‐dimensional correlation spectroscopy, elucidates a sequential response mechanism: the dehydration of hydrophobic methyl groups precedes the rearrangement of amide hydrogen bonds, thereby driving aggregation. This work establishes a synthesis platform for cellulose‐g‐peptoid graft polymers and clarifies the molecular‐to‐macroscopic design principles for thermally triggered gelation.

Thermoresponsive carboxymethyl cellulose (CMC) graft polymers are synthesized via Ugi multicomponent polymerization using two distinct grafting strategies. A reversible sol–gel transition is exclusively observed for polymers with short, dense grafts, whereas long, sparse grafts cause particle aggregation—demonstrating that graft architecture precisely controls the thermal response.

1. Introduction

The development of intelligent responsive materials, particularly thermosensitive polymers capable of sensing and responding to external temperature variations, has garnered widespread attention owing to their immense potential in biomedical fields such as controlled drug release, tissue engineering, injectable scaffolds, and 3D bioprinting [1, 2, 3, 4]. These materials undergo reversible physical or chemical transitions near a specific temperature threshold, enabling precise modulation of cellular microenvironments and drug release kinetics [5, 6]. To date, the most extensively studied thermosensitive polymers include poly(N‐isopropylacrylamide) (PNIPAM) and its derivatives, polyethers such as Pluronic F127, and certain natural polypeptides [7, 8]. Despite exhibiting sensitive and tunable thermoresponsive behavior, these synthetic polymers face inherent limitations in practical applications:  they may be difficult to degrade in vivo or their degradation products exhibit cytotoxicity, while natural peptide‐based systems suffer from inherent instability toward proteases, severely compromising their bioavailability [9, 10].

Peptoids, N‐substituted glycine oligomers, constitute an important class of non‐natural peptidomimetics that offer a promising solution to overcome these challenges [10, 11]. Compared with natural peptides, peptoids not only retain favorable biocompatibility but also exhibit remarkable enzymatic stability and programmable sequence specificity [12, 13]. In recent years, researchers have begun to explore the potential of peptoids for constructing thermosensitive materials. For instance, Samad et al. synthesized a series of peptoid compounds from natural dipeptides via the Ugi multicomponent reaction, preliminarily demonstrating the thermoresponsive capability of such polymers [11]. Nevertheless, fully realizing the potential of peptoids as smart thermoresponsive materials requires addressing two core challenges. First, the thermally induced phase transition of many synthetic peptoids in aqueous solution tends to result in uncontrolled precipitation or abrupt macroscopic phase separation [10], rather than forming a stable, homogeneous hydrogel network suitable for cell encapsulation or drug delivery [14, 15]. Second, the Ugi reaction, while a powerful tool for constructing peptoids, remains underexplored regarding how its reaction conditions precisely regulate peptoid molecular weight, sequence distribution, and topological architecture, and consequently how these parameters govern the resulting macroscopic thermoresponsive behavior and phase transition mechanism.

In response to these challenges, integrating peptoids with a hydrophilic natural polysaccharide backbone has been widely recognized as an ideal strategy for constructing novel bio‐based intelligent materials [16, 17]. This approach fully leverages the synergistic advantages of both components [18, 19]: the polysaccharide framework provides a green, robust, and readily modifiable hydrophilic scaffold that imparts excellent biocompatibility, degradability, and mechanical properties, while the grafted peptoid side chains contribute sensitive stimulus‐responsiveness. Crucially, the highly hydrophilic long‐chain macromolecular backbone of the polysaccharide forces the peptoid segments to undergo constrained intermolecular hydrophobic association [20, 21]. Such restricted hydrophobic aggregation tends to induce the formation of a uniform, physically cross‐linked network that percolates throughout the entire system, thereby establishing the structural foundation for achieving a reversible sol‐gel transition [22].

However, despite the highly attractive concept of combining polysaccharides with peptoids, systematic studies on the construction of thermoresponsive peptide‐polysaccharide hybrids via in situ grafting of peptoids onto natural polysaccharide backbones through the Ugi reaction have yet to be reported. A particularly salient technical bottleneck is the propensity for cyclization side reactions during Ugi polymerization [23, 24]. This side reaction consumes active end groups by generating inert cyclic oligomers incapable of further chain propagation or grafting, thereby severely impeding the controlled construction of linear peptoid chains and the desired graft architecture. Consequently, developing a controllable synthetic strategy that effectively suppresses these deleterious cyclization side reactions, and establishing a clear “synthesis‐structure‐property” relationship based on such control, is a critical prerequisite for advancing polysaccharide‐peptoid smart biomaterials toward practical application. Notably, this prerequisite, namely the controlled suppression of cyclization side reactions and the establishment of well‐defined synthesis‐structure‐property relationships, has also been easily overlooked in existing studies on cellulose‐based thermogels [25, 26], polysaccharide‐grafted thermoresponsive polymers [27, 28], and Ugi‐derived peptoid materials [29, 30].

Herein, Ugi polymerization of glycylglycine (Gly‐Gly) on a carboxymethyl cellulose (CMC) backbone was used to systematically study how reaction conditions influence the resulting cellulose‐g‐peptoid graft polymer structures. Two synthetic strategies, stepwise and one‐pot, were employed to fabricate cellulose‐g‐peptoid graft polymer with tailored architectures (Figure 1). The stepwise method (Scheme 1) enables the tailored regulation of graft length and density, whereas the one‐pot approach (Scheme 2) allows in situ peptoid formation and grafting directly on cellulose, offering operational simplicity and more uniform surface coverage. The chemical structures and thermoresponsive optical properties (cloud points) of the resulting materials were characterized, and the link between graft architecture and macroscopic rheological behavior was investigated. Furthermore, through variable‐temperature infrared spectroscopy and two‐dimensional correlation analysis, we elucidate the molecular‐level interactions governing the thermal response. This work establishes a cellulose‐g‐peptoid graft polymer and systematically discusses how the graft architecture regulates the sol–gel transition, highlighting the essential role of the polysaccharide backbone in achieving reversible gelation.

FIGURE 1.

Schematic diagram of two synthesis strategies for cellulose‐g‐peptoid graft polymer. (Gly‐Gly, glycylglycine; P_m. poly (glycylglycine) peptoid; NP_n, n‐propyl poly (glycylglycine) peptoid; CMC, carboxymethyl cellulose; CNP_n, cellulose‐g‐NP_n; CP_x, cellulose‐g‐poly (glycylglycine) peptoid).

2. Experimental Section

2.1. Materials

Glycylglycine (Gly‐Gly) was obtained from Shanghai yuanye Bio‐Technology Co., Ltd. (Shanghai, China). Carboxymethyl cellulose (CMC, DS = 1.2, M _w = 250, 000 g/mol), formaldehyde (HCHO, 37 wt.%), tert‐butyl isocyanide (98%), n‐propylamine (99%), deuterium oxide (D₂O, 99.9 atom% D) and (dimethyl sulfoxide)‐d6 (DMSO‐d6, 99.5 atom % D) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China).

2.2. Synthesis of Poly (glycylglycine) Peptoid (P_m)

In a typical experiment, glycylglycine was first dissolved in deionized water under constant stirring. An equimolar amount of formaldehyde was then added to the solution, and the mixture was equilibrated at either room temperature (25°C) or low temperature (4°C). Subsequently, an equimolar quantity of tert‐butyl isocyanate was introduced to initiate the polymerization reaction. After allowing the reaction to proceed for 2 h, the product was purified by dialysis (100 Da). The purified samples were obtained by freeze‐drying and were designated as “P_m”, where “m” represents the molar amount of glycine used in the synthesis (detailed synthetic parameters are provided in Table S1).

2.3. Synthesis of n‐Propyl Poly (Glycylglycine) Peptoid (NP_n)

The general synthetic procedure for n‐propylamine‐terminated peptoid was carried out by first dissolving glycylglycine in deionized water, followed by the addition of n‐propylamine and an equimolar amount of hydrochloric acid. The reaction mixture was cooled to 4°C before adding formaldehyde under constant stirring, after which tert‐butyl isocyanate was introduced to initiate polymerization. For procedures requiring multiple additions, glycine dipeptide solution, formaldehyde, and tert‐butyl isocyanate were sequentially administered at 30‐min intervals. Following a total reaction time of 2 h, the product was purified through dialysis (100 Da) and subsequently lyophilized to obtain the final product. The synthesized compounds were designated as “NP_n”, where “n%” represents the molar percentage of n‐propylamine relative to glycylglycine (detailed synthetic parameters are provided in Table S2).

2.4. Preparation of Cellulose‐g‐NP_n (CNP_n)

After the synthesis of NP_n, the undialyzed NP_n was added to a CMC solution under continuous stirring in an ice‐water bath. Subsequently, formaldehyde and tert‐butyl isocyanate were introduced at the same molar ratio as n‐propylamine, and the reaction proceeded for 12 h. The product was dialyzed in water for 24 h using a membrane with a molecular weight cutoff of 8000 Da, followed by lyophilization to obtain the sample. The lyophilized sample was washed with ethanol, and the final mass of the dried product was recorded. The samples were designated as “CNP_n,” where “n%” represents the molar percentage of n‐propylamine relative to glycine. The detailed reaction dosages are provided in Table S2.

2.5. Preparation of Cellulose‐G‐Poly (Glycylglycine) Peptoid (CP_x)

Glycylglycine was first dissolved in a CMC solution, followed by the addition of hydrochloric acid and formaldehyde. The reaction mixture was then cooled to 4°C, and tert‐butyl isocyanate was added to initiate the reaction. For procedures requiring multiple additions, the glycylglycine solution, formaldehyde, and tert‐butyl isocyanate were sequentially introduced at 30‐min intervals, with a total reaction duration of 12 h. The resulting product was dialyzed against water for 24 h using a membrane with a molecular weight cutoff of 8000 Da, followed by lyophilization. The obtained sample was washed with ethanol, and the final mass of the dried product was recorded. The samples were designated as “CP_x,” where “x” denotes the molar percentage of glycylglycine relative to the glucose units of cellulose. Detailed reaction dosages are listed in Table S3.

2.6. Characterization

¹H NMR spectra were obtained using a 600 MHz superconducting nuclear magnetic resonance spectrometer (AVANCE III HD 600, Bruker, Germany). 10 mg of the sample was fully dissolved in 0.5 mL of D₂O or DMSO‐d₆ and transferred to the nuclear magnetic tube for a ¹H spectrum test (scanning 16 times). Diffusion‐ordered spectroscopy (DOSY) spectra were obtained using the same 600 MHz superconducting nuclear magnetic resonance spectrometer (AVANCE III HD 600, Bruker, Germany) with a stimulated echo sequence using bipolar gradient pulses (ledbpgp2s). The gradient strength was linearly ramped from 2% to 95% of the maximum gradient strength (50 G/cm) in 16 steps. The diffusion delay (Δ) was set to 100 ms, and the gradient pulse length (δ) was 2 ms. The temperature was maintained at 298 K. Variable‐temperature ¹H NMR spectra were obtained using the same 600 MHz superconducting nuclear magnetic resonance spectrometer (AVANCE III HD 600, Bruker, Germany) over a temperature range of 25 °C–60 °C with an increment of 5 °C. The sample was equilibrated for 5 min at each temperature before data acquisition. The temperature was controlled with an accuracy of ±0.1 K. XPS spectra were recorded with x‐ray photoelectron spectroscopy (K‐Alpha, Thermo Scientific, America), and the AIKα radiation (1486.6 eV) was selected as an excitation source. The molar mass was determined based on gel permeation chromatography measurement (1260 Infinity II, Agilent, America). DMF was used as the mobile phase for testing, and 0.2 wt.% P_m and NP_n solutions were filtered through a 0.22 µm filter membrane before testing. The elemental analysis, including carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), was performed with an elemental analyzer (Vario EL cube, Elementar, Germany). Each sample was measured at least twice. Electrospray ionization mass spectroscopy (ESI‐MS) was performed on a high‐resolution mass spectrometry (1290‐6545XT, Agilent, America) in the positive ion mode. Turbidity was determined by measuring the absorbance at 260 nm on a circular dichroism spectrum (Chirascan, Applied Photophysics, UK), with the temperature maintained at a constant rate of 2°C/min. Dynamic light scattering (DLS) was used to obtain the size distribution of the particles by using a nanoparticle size potentiometer (ZTS1240, Malvern Panalytical, UK) at 45 °C. The rheology was investigated using a multi‐function rotary rheometer (Anton Paar, Austria), and the flat plate (25 mm) was selected as the measurement system. The measurement condition was a 2°C/min heating rate and a 1 Hz oscillation frequency. The zeta (ζ) potential is measured through a nanoparticle size potentiometer. The FTIR spectra (Nicolet iS50, Thermo Scientific, America) were recorded, equipped with a DTGS detector. Two pieces of microscope CaF₂ windows, which have no absorption bands in the Mid‐infrared region, were used to prepare a transmission cell. Variable‐temperature spectra were collected between 20 and 40°C with an increment of 1°C (accuracy:  0.5°C). The manual method was used to change the temperature; each temperature point was maintained for 30 min to collect the IR spectrum, and then the temperature was increased or decreased by 1°C manually. The baseline correct processing was performed by the software of OMNIC 8.0, and the mapping was completed by the 2D Shige software.

2.7. Calculation Methods of Grafting Density φ

The grafting density φ of each product was calculated based on the nitrogen content of the sample (the derivation of equation was recorded in SI), using the following equation:

$$\phi =\frac{M_{CMC}\times N\%}{3M_N-\left(M_{Ugi}\times N\%\right)}÷\mathrm{DP}\left(N{P}_n\right)$$ (1)

where φ represents the quantity ratio of the grafted polypeptide chain to the cellulose glucose units, M_CMC was the molar weight of one anhydroglucose unit containing carboxyl groups (231.81 g/mol), N% was the mass concentration of nitrogen, M_N was the molar weight of one atom of nitrogen (14.01 g/mol), M_Ugi was the molecular weight of the characteristic Ugi reaction product structure, (227.3 g/mol), $\mathrm{DP}(N{P}_n)$ was derived from the average molecular weight of the peptoid chain (NP_n) and the molecular weight of a single repeat unit (M_Ugi )

3. Results and Discussion

3.1. Preparation and Characterization of Poly (Glycylglycine) Peptoid (P_m)

Glycylglycine (Gly‐Gly), as the simplest dipeptide, is first considered in this study. It is dissolved in water, followed by the sequential addition of formaldehyde and tert‐butyl isocyanide (detailed in Table S1). The reaction is allowed to proceed for 2 h at either room temperature (25°C) or low temperature (4°C). The structures of the starting material and the resulting poly (glycylglycine) peptoid (P₅) are confirmed by ¹H NMR analysis. As shown in Figure 2a, the characteristic signals at 3.58 ppm and 3.64 ppm (labeled a₁) correspond to the two methylene groups of glycylglycine. The characteristic signal at 8.15 ppm (b₁) represents its amide group. In the spectrum of P₅, the characteristic signal at 1.27 ppm (a₂) is assigned to the methyl groups, the signals in the region of 3.75–4.25 ppm (b₂) to the methylene groups, and the signals in the region of 8.50–9.00 ppm (c₂) to the amide groups. Moreover, the occurrence of the Ugi reaction is further verified by XPS. The survey spectrum of P₅ (Figure 2b) shows a significantly enhanced carbon peak compared to that of the starting material, indicating successful incorporation of carbon‐rich components after the reaction. Furthermore, the high‐resolution C 1s spectrum (Figure 2c) indicates that this change primarily resulted from a substantial increase in the proportion of C─C bonds. This finding confirms the successful grafting of the tert‐butyl group from tert‐butyl isocyanide onto the polymer via the Ugi reaction.

FIGURE 2.

(a) The ¹H NMR spectra of Gly‐Gly and P₅. (b) The survey spectrum of Gly‐Gly and P₅. (c) The high‐resolution C 1s spectrum of Gly‐Gly and P₅.

3.2. Synthetic Reaction Characteristics of P_m

The characteristics of the polymers synthesized via the Ugi reaction are investigated by GPC, with the results summarized in Table 1. By comparing the molecular weights obtained at different reactant concentrations, it is evident that an increase in concentration favors the Ugi reaction. Specifically, polymer P_1‐1 (Mw = 9500 g·mol⁻¹, Mn = 3800 g·mol⁻¹) exhibits higher molecular weights than P_1‐2 (Mw = 7700 g·mol⁻¹, Mn = 3200 g·mol⁻¹), with average degrees of polymerization increasing by 8.1 and 2.6, respectively. Furthermore, while maintaining a constant reactant concentration, the molar amounts of reagents (glycylglycine/formaldehyde/tert‐butyl isocyanide) per batch are incrementally increased (1/1/1 mmol, 5/5/5 mmol, 25/25/25 mmol, 50/50/50 mmol). The molecular weights (Mw, Mn) of the resulting polymers initially increase and then decrease. This trend is primarily attributed to the exothermic nature of the Ugi reaction. At lower molar amounts (1/1/1 mmol, 5/5/5 mmol), the heat released is insufficient to significantly raise the system temperature. In this regime, a higher reagent quantity per batch promotes an increase in molecular weight and a reduction in the polydispersity index (PDI_1/1/1 = 2.51; PDI_5/5/5 = 1.98), indicating enhanced polymerization efficiency and a decrease in low‐molecular‐weight oligomers. However, when the molar amount per batch is further increased, the exothermic effect becomes non‐negligible due to the accelerated reaction in aqueous solution [31]. The resulting temperature rise reduces reaction efficiency, leading to a decline in polymer molecular weight. Additionally, comparing polymers synthesized at different temperatures (4°C and 25°C) reveals that lower reaction temperatures yield higher molecular weights, demonstrating that a cooled environment improves the efficiency of the Ugi reaction. In summary, these results indicate that managing heat release and controlling the ambient temperature are crucial factors within the Ugi reaction system for optimizing polymerization outcomes.

TABLE 1.

The molecular weights of various poly (glycylglycine) peptoids.

Polymer	Gly‐Gly/HCHO/tBuNC (mmol)	H2O (mL)	Temperature (°C)	Mw (g·mol−1)	Mn (g·mol−1)	Ð
P1‐1	1/1/1	0.6	25	9500	3800	2.51
P1‐2	1/1/1	3.0	25	7700	3200	2.40
P5	5/5/5	3.0	25	9700	4900	1.98
P25‐1	25/25/25	15	25	7600	4400	1.72
P25‐2	25/25/25	15	4	8200	4800	1.70
P50	50/50/50	30	25	6300	2600	2.44

In the Ugi multicomponent reaction, the resulting polymers inherently possess both amino and carboxyl groups, making the side reaction “ring‐closing reaction” inevitable. This leads to the formation of cyclic oligomers (Figure 3, polymer A), which lack functional groups such as carboxyl and amino groups and are therefore unfavorable for subsequent grafting onto polysaccharide chains. The “capping” strategy, which involves adding a monoamine or monocarboxylic acid to terminate the polymer ends and prevent cyclization, effectively addresses this issue. The product structure obtained via this strategy differs significantly from that of the “non‐capped” approach. In this study, n‐propylamine was selected as the ring‐closing inhibitor. Electrospray ionization (ESI) analysis was performed to compare the products obtained without capping (poly (glycylglycine), P₂₅) and with n‐propylamine capping (n‐propyl poly (glycylglycine) peptoid, NP₅).

FIGURE 3.

The ESI analysis of P₂₅ and NP₅.

As shown in Figure 3, P₂₅ displays signals corresponding to both cyclic oligomers (polymer A) and non‐cyclic polymers (polymer B). The relatively higher intensity of the cyclic oligomer signals, although such cyclic species lack the amino and carboxyl groups that typically enhance ionization efficiency in ESI, strongly implies that cyclic oligomers constitute a substantial fraction of the uncapped product. In stark contrast, the n‐propylamine‐capped product NP₅ exhibits a dramatic reduction in cyclic oligomer signals alongside an increase in non‐cyclic polymers (polymer B). Furthermore, a new species corresponding to n‐propylamine‐capped non‐cyclic polymer (polymer C) is observed. This spectral shift provides direct evidence that the capping strategy effectively suppresses the ring‐closure side reaction, yielding a product with a higher proportion of graftable linear species. Additionally, ESI results from the “one‐pot” (NP_5‐1) and “stepwise‐addition” (NP_5‐2) procedures (Figure S1) show that the cyclic polymer content of NP_5‐1 is higher than that of NP_5‐2, indicating that moderately increasing the proportion of n‐propylamine can inhibit the “cyclic closure side reaction”.

3.3. Preparation and Characterizations of Cellulose‐g‐Poly (Glycylglycine) Peptoid (CP_x) and Cellulose‐g‐NP_n (CNP_n)

The chemical structures of the obtained thermosensitive cellulose graft products (CP_x and CNP_n) were characterized by a combination of techniques. ¹H NMR spectroscopy (Figure 4 and Figures S2 and S3) provides supporting evidence for the presence of both cellulose backbone and peptoid components. Figure 4 displays the ¹H NMR spectra of CMC, CP_2.5, and CNP₈₀. First, the broad peak a₁ in the range of 3.00–4.50 ppm corresponds to the protons on the sugar ring of the cellulose backbone, clearly demonstrating the polysaccharide component in both CP_2.5 and CNP₈₀. Furthermore, the characteristic signal b₂ in the region of 3.75–4.25 ppm is assigned to the methylene groups of the peptoid‐based polymer. A pronounced signal peak a₂ appears at 1.25 ppm, belonging to the methyl protons of the tert‐butyl groups in the peptoid, which reflects the polypeptoid component in CP_2.5 and CNP₈₀. Additionally, due to the introduction of the n‐propylamine moiety in CNP₈₀, distinct signals corresponding to the n‐propyl group (a₃, b₃, c₃) are readily observed in its spectrum. Finally, comparisons of the graft products with different grafting ratios within the CP_x and CNP_n series (Figures S2 and S3) show that all samples exhibit similar characteristic signals, supporting the presence of the grafted structure.

FIGURE 4.

The ¹H NMR spectra of CMC, CP_2.5, and CNP₈₀.

Notably, direct quantitative verification of the graft architecture (chain length and grafting density) on the intact graft polymer by ¹H NMR spectroscopy is hindered by the broad signals of the cellulose backbone and their overlap with the signals of the peptoid chains. The method for directly characterizing the graft structure is currently unclear. Therefore, for the CP_x series, the structure cannot be directly inferred; for the CNP_n series, however, the chain length and grafting density can be indirectly estimated using the molecular weight of the peptoid chains independently characterized before grafting. Nevertheless, it is first necessary to confirm the absence of low molecular weight impurities (e.g., unreacted peptoid oligomers) in the graft products. The diffusion coefficient data from DOSY NMR of CNP₂₀ and CNP₈₀ indicate a single diffusing component (Figure S4); no signals corresponding to free peptoid oligomers (which would exhibit distinct diffusion coefficients) were detected. This confirms that unreacted or physically mixed peptoid species have been completely removed by the purification protocol. Therefore, the graft length and grafting density of the CNP_n series can be indirectly calculated by combining the degree of polymerization of the peptoid chains (Table S4).

However, it is important to note that, even with this indirect validation, the current synthetic approach does not permit the independent variation of graft length and density, as these parameters are coupled through the reaction conditions. Specifically, a higher capping agent content leads to lower molecular weight peptoids (e.g., NP₈₀) and a greater number of active chain ends (amine groups), thereby constructing short and dense graft polymers (CNP₈₀). Conversely, reducing the capping agent content increases the peptoid molecular weight (e.g., NP₂₀) and reduces the number of active ends, favoring the formation of long and sparse graft polymers (CNP₂₀). Moreover, although the capping strategy described in Section 3.2 demonstrably reduces cyclization, it does not eliminate cyclic byproducts entirely, and we currently lack a method to separate cyclic from linear grafted species. Consequently, the estimated graft architecture for the CNP_n series may still contain unquantified contributions from cyclic structures. The comparisons among CNP₅, CNP₂₀, and CNP₈₀ involve samples that differ in multiple parameters simultaneously. Accordingly, the observed thermoresponsive behavior is correlated with the overall graft architecture, rather than the isolated effect of a single parameter such as graft length or grafting density. This limitation must be clearly explained in the current work.

3.4. Temperature‐Dependent Optical Properties of P_m, CP_x, and CNP_n

The thermosensitivity of these graft products in aqueous solution was further investigated after structural confirmation. Both the polypeptoid polymer (P₅) synthesized via the Ugi reaction of glycylglycine and the cellulose grafted derivatives (CNP₅, CP₂₀) exhibit thermoresponsive behavior in water (Figure 5a), a valuable property for certain biomedical applications. Figure 5b shows the thermoresponsive behavior of a 1 wt.% P_m solution with different molecular weights during heating. As shown by the turbidity curves, the polymer solution with a higher molecular weight (P₅) displays a lower cloud point temperature (T_cp). This is likely because higher molecular weight polymers tend to form larger aggregates more readily upon heating, which results in earlier detection of turbidity. Furthermore, by comparing the turbidity curves of P₅ solutions at different concentrations during heating (Figure 5c), a concentration dependence of T_cp is observed, with higher concentrations resulting in a lower T_cp. The thermoresponsive performance of cellulose grafted with varying‐molecular‐weight polypeptoid chains was also studied. Figure 5d reveals that the T_cp is primarily determined by the molecular weight of the grafted polypeptoid side chains (Mw: CNP₅ = 9300 g·mol⁻¹, CNP₁₀ = 8700 g·mol⁻¹, CNP₂₀ = 6100 g·mol⁻¹): a higher molecular weight of the polypeptide leads to a lower T_cp of the corresponding CNP. Additionally, the nitrogen content of the product can be used as a comprehensive parameter to reflect the grafting density and chain length (Table S4). At the same CNP concentration, the ultimate absorbance of the CNP solution shows a clear positive correlation with this parameter. Dynamic light scattering measurements at 45°C (Figure 5e) indicate that CNP₁₀, with the highest repeat unit proportion, exhibits the largest particle size, while CNP₂₀, with the lowest proportion, shows the smallest size. Therefore, increasing the proportion of repeat units promotes the formation of larger aggregates by CNPs at elevated temperatures, resulting in an enhanced ultimate absorbance of the CNP solution under heating.

FIGURE 5.

(a) The changes of P₅, CNP_5, and CP₂₀ solutions (2 wt%) at different temperatures. (b) The turbidity changes curves of P₅, P_25, and P₅₀ solutions (1 wt%) at different temperatures. (c) The turbidity changes curves of P₅ solutions with different concentrations at different temperatures. (d) The absorbance changes curves of CNP₅, CNP₁₀, and CNP ₂₀ solutions (2 wt.%) at different temperatures. (e) Dynamic light scattering measurements of CNP ₅, CNP₁₀, and CNP ₂₀ solutions (0.1 wt%) at 45°C.

3.5. Influence of Peptoid Grafting Strategy on Thermoreversible Gelation

The thermal responsiveness of materials is typically reflected in both optical and rheological properties. The effects of different polypeptide grafting strategies on the thermosensitive rheological behavior of cellulose were further investigated using four thermosensitive cellulose samples with similar grafting contents (nitrogen content: 12.0%–12.7%). As shown in Figure 6a, all four samples exhibit viscous flow behavior dominated by energy dissipation similar to a fluid at low temperature (5°C). However, when the temperature reaches the physiological range, only the CNP_{80 (12.0)} solution undergoes a sol‐gel transition, while the other three systems remain in a solution state without significant change. Subsequently, changes in absorbance were examined. Figure 6b shows that the absorbance of CNP_{80 (12.0)} begins to increase first, likely because its thermoresponsive grafts are more dispersed, facilitating aggregation and light scattering upon heating. The zeta potentials of the raw material CMC and the four grafted cellulose samples were then measured (Figure 6c). CMC exhibits the highest zeta potential due to its abundant carboxyl groups. For the CNP series, the zeta potential gradually decreases as the grafted chains become shorter, which can be attributed to the consumption of more carboxyl sites during grafting. Although CP_{10 (12.4)} possesses uncapped polypeptide side chains, its significantly reduced zeta potential is likely due to the electrostatic shielding effect exerted by the densely grafted chains on the surrounding non‐grafted carboxyl groups.

FIGURE 6.

(a) The changes of 2 wt% CNP_{80 (12.0)}, CNP_{5 (12.7)}, CNP_{20 (12.3),} and CP_{10 (12.4)} solutions at different temperatures. (b) The absorbance change curves of 2 wt% CNP_{80 (12.0)}, CNP_{5 (12.7)}, CNP_{20 (12.3),} and CP_{10 (12.4)} solutions at different temperatures. (c) The zeta potential of CNP_{80 (12.0)}, CNP_{5 (12.7)}, CNP_{20 (12.3),} and CP_{10 (12.4)} solutions. (d) The energy storage modulus G’ and loss modulus G″ of CNP_{80 (12.0)} with different concentrations in the range of 10°C–60°C. (e) The phase states of CNP_{80 (12.0)} at various concentrations and temperatures.

Furthermore, the temperature‐induced sol‐gel transition of CNP_{80 (12.0)} was studied in detail by rheological measurements. The gelation temperature was defined as the temperature at which the storage modulus (G') equals the loss modulus (G'') during a temperature sweep. As shown in Figure 6d and Figure S5, the thermoresponsive rheological behavior of CNP_{80 (12.0)} was tested at different concentrations. At a concentration of 1 wt.%, the solution transitions to a gel at 24.7°C, while at 2 wt.%, gelation occurs at 20.3°C. The phase states of CNP_{80 (12.0)} at various concentrations and temperatures are summarized in Figure 6e, which clearly delineates its sol‐gel transition region. As the concentration increases, the gelation temperature of CNP_{80 (12.0)} gradually decreases, reaching as low as 20°C. To further verify this gel‐like behavior and its reversibility, additional rheological experiments were performed. Using 2 wt.% CNP_{80 (12.0)} as a representative sample, strain sweep measurement was first carried out to determine the linear viscoelastic region, where the rheological parameters remained stable over a strain range of 0.1%–100% (Figure S6a, f = 1 Hz, T = 37°C). Frequency sweep measurement performed at 37°C and 1% strain shows that both G' and G'' remained constant at frequencies below 10 Hz (Figure S6b), confirming the formation of a stable gel network. Finally, a heating‐cooling cycle rheological test was performed by alternating the temperature between 25°C and 37°C at a rate of 5°C/min (Figure S6c). Over three complete cycles, G' and G'' were almost fully recovered during each cooling stage, indicating that the sol‐gel transition of CNP_{80 (12.0)} is completely thermoreversible.

To further distinguish between genuine network formation and simple particle aggregation, we performed concentration‐dependent dynamic light scattering measurements on the representative the non‐gelling sample CNP₂₀ and gelling sample CNP_{80 (12.0)} (Figure 7). For CNP₂₀, the particle size increased only gradually with concentration, and the size distribution remained unimodal and narrow, indicating that the limited inter‐particle aggregation, with aggregate sizes confined to a relatively small range. In contrast, the CNP_{80 (12.0)} shows a significant widening of the particle size range at the critical concentration (0.8wt%), indicating a transition from isolated chains (or chain clusters) to a macroscopic network. These results demonstrate that CNP₂₀ and CNP_{80 (12.0)} follow fundamentally different aggregation pathways: long and sparse grafts lead to particle‐like aggregates, whereas short and dense grafts promote the formation of an interconnected network structure.

FIGURE 7.

(a) Dynamic light scattering measurements of CNP₂₀ and (b) CNP_{80 (12.0)} solutions with different concentrations at 45°C.

3.6. The Temperature‐Sensitive Mechanism of Peptides and Thermosensitive Cellulose

To further elucidate the thermal response mechanism of the polymer, in situ infrared spectroscopy was employed to monitor spectral changes during the heating of a 20 wt.% P₅ solution in D₂O. D₂O was selected over H₂O as the solvent to eliminate the interference from the bending vibration absorption of water molecules near 1640 cm⁻¹ [32]. As shown in Figure 8a, upon heating, the methyl vibrational band ν(CH₃) at 2970 cm⁻¹ shifts to lower wavenumbers. This red shift can be attributed to the transition of CH₃ groups from a hydrated state with weak interactions with water molecules to a hydrophobic microdomain environment where they cluster together. The weakened intermolecular interactions reduce the energy required for the stretching vibration, resulting in the observed red shift of the methyl peak. Furthermore, Figure 8b shows that the carbonyl stretching vibration ν(C═O) of the amide bond at 1620 cm⁻¹ shifts to higher wavenumbers with increasing temperature, exhibiting a blue shift. This is due to the conformational transition of P₅ chains from a coil to a globular state during heating. As the temperature rises, the original hydrogen bonds between the carbonyl groups and D₂O solvent (─C═O···D‐O‐D) are disrupted. Concurrently, stronger intramolecular or interchain hydrogen bonds (─C═O···D‐N‐) form, enhancing electron delocalization in the C═O bond and increasing its force constant [33]. Consequently, the stretching vibration frequency increases, leading to the observed blue shift.

FIGURE 8.

(a,b) FTIR spectra of 20 wt.% P₅ solution in D₂O during the heating process. (c) Synchronous and (d) asynchronous maps of 20 wt.% P₅ solution in D₂O in the regions 1500–1660 and 3020‐2860 cm⁻¹ during the heating process.

Since the structural changes of C‐H and amide groups during heating are difficult to discern directly from the original spectra, the temperature‐dependent spectral dataset was subjected to hetero two‐dimensional correlation spectroscopy analysis. The synchronous map (Figure 8c) reveals the homogeneity of spectral signals, while the asynchronous map (Figure 8d) correlates this information with the sequence of events. A distinct negative cross‐peak appears near (2970, 1620 cm⁻¹) in the synchronous map, and the corresponding cross‐peak in the asynchronous map is also negative. This indicates that the signal change at 2970 cm⁻¹ (C‐H) precedes that at 1620 cm⁻¹ (amide group), suggesting that dehydration of the methyl groups occurs first upon heating, followed by the formation of hydrogen bonds involving the amide groups. This result further demonstrates that the influence of temperature on the structure of the polypeptoid material follows a hierarchical sequence: the related structure of C‐H is more sensitive to thermal perturbation, while the amide group exhibits relatively higher stability and a delayed response.

To directly verify whether the grafted peptoid chains retain this sequential thermoresponsive behavior and to confirm that the structure‐dependent gelation originates from the peptoid grafts on the cellulose backbone, we performed variable‑temperature ¹H NMR measurements on P₅, CNP₂₀, and CNP_{80 (12.0)} under identical conditions (Figure 9). In the variable‑temperature ¹H NMR spectra of P₅, the intensities of the characteristic peaks a₁ and b₁ progressively decrease with increasing temperature, which can be attributed to phase separation and precipitation of the free peptoid. Notably, the characteristic peptoid proton signals in both CNP₂₀ and CNP_{80 (12.0)} likewise diminish gradually upon heating, closely mirroring the trend observed for free P₅. This result confirms that the thermoresponsive behavior of the grafted cellulose system similarly originates from the grafted peptoid chains.

FIGURE 9.

(a) Variable‑temperature ¹H NMR spectra of P₅, (b) CNP₂₀, and (c) CNP₈₀.

According to the sequence of group changes revealed by the two‐dimensional infrared spectroscopies and the variable‐temperature ¹H NMR spectroscopies, Figure 10a illustrates the molecular mechanism underlying the thermal response of the polymer. With increasing temperature, the polymer undergoes dehydration in a stepwise manner: first, the CH₃ groups at 2970 cm⁻¹ dehydrate, weakening the interactions between the hydrated CH₃ groups and surrounding water molecules. The resulting hydrophobic interactions then drive the alkyl groups to approach and aggregate. Second, the hydrogen bonds between the amide bonds (─C = O, ─N─H) at 1620 cm⁻¹ and the surrounding water molecules begin to break. Third, the number of intramolecular or interchain hydrogen bonds (─C═O···H─N─) increases, further promoting polymer association and aggregation. These sequential changes collectively give rise to the observed thermoresponsive behavior of the polymer.

FIGURE 10.

(a) The molecular mechanism underlying the thermal response of the peptide. (b) The mechanism behind the distinct rheological behaviors exhibited by cellulose grafted with different structures.

Furthermore, Figure 10b explains the mechanism behind the distinct rheological behaviors exhibited by cellulose grafted with different structures. CNP_{80 (12.0)}, characterized by shorter graft chains and a higher grafting density, readily forms interchain associations with neighboring grafted chains. This facilitates the establishment of a cross‐linked network, enabling a temperature‐induced sol‐gel transition. In contrast, CNP₅ and CNP₂₀ possess longer graft chains and a lower grafting density. Here, aggregation primarily occurs within individual grafted polymer chains. This intramolecular aggregation promotes the formation of particle‐like structures upon heating, allowing the solution to largely retain its fluidity and resulting in minimal changes in rheological properties. Moreover, when the molecular weight of the grafted chains increases, the coiled aggregates contain a greater number of intramolecular hydrogen bonds (─C═O···H─N─), favoring the formation of larger particles. On the other hand, CP₁₀, bearing hydrophilic carboxyl groups on its graft chains, experiences strong electrostatic repulsion between molecules in solution. This repulsion inhibits the formation of aggregates. Consequently, the solution remains liquid and shows almost no change in absorbance upon heating.

4. Conclusions

In summary, a strategy is developed for synthesizing thermoresponsive cellulose‐g‐peptoid graft polymers via Ugi multicomponent reaction. The employment of a capping agent and modulation of reaction conditions effectively suppresses cyclization side reactions, enabling the achievement of tailored graft architectures with specific lengths and densities. Additionally, the thermosensitivity is found to be critically governed by this graft architecture. Short, dense grafts (CNP_{80 (12.0)}) promote intermolecular associations, resulting in a sol‐gel transition phenomenon during the heating process. In contrast, longer, sparser grafts (CNP₅, CNP₂₀) favor intramolecular folding and hydrophobic self‐aggregation, resulting in increased turbidity and particle formation without macroscopic gelation. Notably, the introduction of hydrophilic carboxyl groups into the grafts (CP₁₀) leads to strong electrostatic repulsion, which suppresses intermolecular aggregation and thereby diminishes its thermoresponsive behavior. Variable‐temperature FTIR and 2D IR spectroscopy elucidate a sequential molecular mechanism for the transition, which progresses from the dehydration and aggregation of hydrophobic moieties, through the detachment of amide groups from surrounding water molecules, to the final establishment of a new hydrogen‐bond network that drives and stabilizes the macroscopic phase transition. The contribution of this work is the construction of CMC‐g‐peptoid hybrids and the demonstration of how graft architecture regulates the sol‐gel transition. This work offers a design principle for developing bio‐based thermoresponsive hydrogels with tunable gelation behavior.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: marc70362‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.