Chemical Modification

In recent years, several reviews have focused on the biosynthesis, structural analysis, and general biomedical/food applications of curdlan (Cai and Zhang 2017). However, its property of being insoluble in water is unfavourable for many applications.

FIGURE 10.3 Chemical approaches for modification of curdlan.

especially for biomedical applications. This prompted the researchers to explore another way of using this polysaccharide, synthesizing curdlan derivatives with good water solubility as well as biological activity. In this sense, the chapter presents the latest important chemical approaches described in the literature for modifying the curdlan with potential industrial or medicinal importance (Figure 10.3).

Carboxylation

A chemical modification procedure of polysaccharides by substitution reactions is the carboxymethylation, which gives products in which the primary and/or secondary alcohol groups are etherified with carboxymethyl groups.

Curdlan was carboxymethylated in an aqueous alkaline medium using monochlo- roacetic acid as the etherifying agent (Sasaki et al. 1979). The yield of this method was about 95%, and the carboxymethylated curdlan (CMCurd) was a water-soluble derivative (70 mg mb'¹) with degree of substitution about 0.47-0.65 (Jin et al. 2006). Table 10.1 includes some key features of CMCurd.

TABLE 10.1

The Main Characteristic of Carboxymethylated Curdlan

The structure of carboxymethylated curdlan was analysed by FTIR and NMR spectroscopy, which revealed that the carboxymethyl group was introduced mainly at the C-6 position as w'ell as at the C-2 and C-4 positions. Figure 10.4 shows the comparative FTIR spectra of native curdlan and CMCurd, and the specific absoiption bands are summarized in Table 10.2.

The NMR spectroscopy also allowed the confirmation of the carboxylation reaction of curdlan, highlighting the specific signals for each atom: in spectrum of the ^L,C

FIGURE 10.4 FTIR spectra of native curdlan and the carboxymethylated derivative (Jin et al. 2006).

TABLE 10.2

Specific Absorption Bands of Curdlan and Carboxymethylated Derivative

NMR the signals 103.7-96.7 (C-l), 75.1-73.9 (C-2), 86.7-76.7 (C-3), 70.6-68.6 (C-4), 76.6-76.8 (C-5), and 61.3-61.7 (C-6) ppm are attributed to the backbone chain of curdlan. In the spectrum of CMCurd, a new signal appears due to the carboxyl group at about 178 ppm. The distinct increase in signal intensities around 70 ppm is attributed to the shift of primary carbon (C-6) from the region around 60 ppm after substitution with -CHUCOOH group on the primary carbon. CMCurd was also used as parent polymer to synthesized new derivatives such as deoxycholic acid modified CMCurd (Gao et al. 2008).

Shibakami et al. synthesized a carboxylate derivative of curdlan by reaction with succinic anhydride in DMAc/LiCl medium (Shibakami et al. 2013). The reaction took place in a homogeneous medium leading to a white, water-soluble product with a DS value of 0.46.

The carboxylic derivative of curdlan with vinyl groups was synthesized by esterification reaction with maleic anhydride (Popescu et al. 2018). The opening of the anhydride cycle and the free carboxylic group obtained ensured the pH sensitivity of the curdlan derivative. The reaction was performed with maleic anhydride and TEA as catalyst, in DMF/LiCl medium, at 60°C. The maleilated curdlan (DS = 0.8 calculated from ‘H NMR spectrum) was used as macromolecular cross-linker in the polymerization of N-isopropylacrylamide (NIPAM) for obtaining thermo and pH- sensitive networks with high porosity.

Sulfation

The chemical modification of curdlan by the introduction of sulfate groups has been extensively studied due to the new properties conferred by these derivatives, such as anticoagulant, antithrombotic activities. The protocol of the sulfation reaction developed for some insoluble polysaccharides, such as cellulose or starch, was also applied to curdlan. The most used sulfating reagents are summarized in Table 10.3.

Chemical structure of curdlan sulfate (CurdS) and positions of sulfate groups in anhydrogluco pyranose units (AGUs) were extensively studied by FTIR and NMR spectroscopy ('H NMR. ^I3C NMR. 2-D correlation experiments). The sulfation reactions take place almost exclusively at primary OH (C-6) when lower values of DS are obtained and then at secondary OH (C-2) when DS is increased. A very little substitution at OH (C-4) was reported in case when DS > 1.6. Compared with the FTIR spectra of curdlan (see Figure 10.4), new adsorption peaks appeared at 870-690 cm^-1 attributed to symmetrical C-S-0 stretching, and 1260-1200 cm⁴, 1070-1030 cm^-1 represent the asymmetrical and symmetrical S=0 stretching vibrations from R-SO,^- groups in CurdS (Pretsch et al. 2010; Li et al. 2014; Jin et al. 2020).

The ^L,C-NMR spectrum of curdlan sulfate recorded in DMSO-d₍, at 60°C, evidenced three new signals at C-6s (67.9 ppm), C-2s (86.0 ppm), and C-4s (79.0 ppm) which were attributed at sulfated groups from C-6, C-4, and C-2 positions of AGU.

The molecular weight (M„.) and intrinsic viscosity [//] of sulfated derivatives were determined by multi-angle light scattering and viscosimetry. The M„ value (2.4 x 10⁴) of sulfated derivatives was much lower than parent curdlan (44.5 x 10⁴) which indicates a degradation of the polymer chain during the reaction. The Mark-Houwink equation and average value of characteristic ratio С_ю (С_ю = 16) in 0.2 M NaCl

TABLE 10.3

The Main Sulfating Agents of Curdlan and the Characteristics of the Derivatives

aqueous solution at 25°C was found to be [q] = 1.32x KT'M,'⁰⁶ (cm-¹ g^_l) (Zang et al. 2000). The 1.06 value of exponent of the equation indicates that the sulfated curdlan has more extended polymeric chains than original curdlan, where the [q] - M„ relationship was given to be [q] = 71 x 10 cm³ g~', due to the electrostatic

repulsion of sulfate groups.

Curdlan sulfate has also been used as parent polymer in different derivatization syntheses such as acetyl curdlan sulfate (Osawa et al. 1993), azidothymidine curdlan sulfate (Gao et al. 1998), O-palmitoyl curdlan sulfate (Lee et al. 2005; Cremer et al. 2010), 6-amino-6-deoxy curdlan sulfate (Borjihan et al. 2003).