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Würfel, H., Geitel, K., Qi, H., and Heinze, T. (2021). "Chemical modification of pectin and polygalacturonic acid: A critical review," BioResources 16(4), Page numbers to be added.

Abstract

Pectin, as a sustainable biopolymer with its two complementary functionalities (carboxyl and hydroxyl moieties) imparted in the α-1,4-galacturonic acid repeating unit, has gained increasing attention in the last few years. The interest in this ubiquitously occurring plant originating polysaccharide (PS) has shifted slowly from applications as a food additive to a broader range of potential applications in medicine, cosmetics, and other industries. Due to the increasing interest in alternatives for petrochemical materials, PSs as biomaterials have gained increasing attention in industrial processes in general. In the last decade, an increasing number of chemical transformations related to pectin have been published, and this is a prerequisite for the design of the structure and hence properties of novel biopolymer-based materials. This work aims to review the chemical modifications of pectin by covalent linkage of the last decade and analyze the materials obtained with these chemical methods critically.


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Chemical Modification of Pectin and Polygalacturonic Acid: A Critical Review

Hendryk Würfel,a Katja Geitel,a Haisong Qi,b and Thomas Heinze a,*

Pectin, as a sustainable biopolymer with its two complementary functionalities (carboxyl and hydroxyl moieties) imparted in the α-1,4-galacturonic acid repeating unit, has gained increasing attention in the last few years. The interest in this ubiquitously occurring plant originating polysaccharide (PS) has shifted slowly from applications as a food additive to a broader range of potential applications in medicine, cosmetics, and other industries. Due to the increasing interest in alternatives for petrochemical materials, PSs as biomaterials have gained increasing attention in industrial processes in general. In the last decade, an increasing number of chemical transformations related to pectin have been published, and this is a prerequisite for the design of the structure and hence properties of novel biopolymer-based materials. This work aims to review the chemical modifications of pectin by covalent linkage of the last decade and analyze the materials obtained with these chemical methods critically.

Keywords: Pectin; Polygalacturonic acid; Chemical modification; Alkylation; Acylation; Amide; Ester; Hydrazide; Polysaccharide; Hydrogel

Contact information: a: Center of Excellence for Polysaccharide Research, Institute of Organic Chemistry and Macromolecular Chemistry, Friedrich-Schiller-University of Jena, Jena, Germany;

b: State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China;

* Corresponding author: Tel: +49 3641 948270, Email: thomas.heinze@uni-jena.de

INTRODUCTION

Pectin, as a plant-based polysaccharide (PS), occurs predominantly in the primary cell wall and the middle lamella and maintains the structural integrity of the cell and provides adhesion between them (Mohen 2008). It was first mentioned in 1790 by Vauquelin, who isolated the material from tamarind and got its name finally in 1825 (Ropartz and Ralet 2020). Due to its water solubility, the PS can be extracted by a water-based procedure under neutral pH conditions; however, acidic extraction procedures increase the extraction efficiency dramatically. The structural elucidation of extracted pectin is a laborious and time-consuming procedure even today, due to a large number of neutral sugars imparted (Morris and Binhamad 2020). The material can be considered as a hetero-polysaccharide consisting mainly of -1,4-galacturonic acid chains, which are esterified at the C6-position with a methyl moiety to a varying extent (Fig. 1).

Fig. 1. (A) Main repeating units of pectin consisting of α-1,4-glycosidic linked galacturonic acid. Esterification at C6 with methyl moieties and acetylation at C2 and C3 to a varying degree, depending on the polysaccharide source, is observed. (B) Arabinan side chain typically occurring in sugar beet shows acylation with ferulic acid to a varying degree

Additionally, the main chains can be interrupted by -1,2-linked L-rhamnose units, which may possess neutral sugars attached as well. The main features found in naturally occurring pectin are homogalacturonan, rhamnogalacturonan I and II, xylogalacturonan, and apiogalacturonan. These structural features can be summarized with the term multi-block co-biopolymer (Ropartz and Ralet 2020). The predominant homoglacturonan structure can be found as the methyl ester to different degrees. A pectin with less than 50% of galacturonic acid unit esterified is considered low-methoxy pectin (LMP), whereas with more than 50% degree of esterification it is high-methoxy pectin (HMP) (Seymour and Knox 2002). The degree of esterification at the C6 position has a large influence on the ability to form gels. HMP can form gels at low pH values in the presence of high concentration of neutral sugars. This property is utilized for the preparation of fruit jellies and jams. LMP forms gels with multivalent metal ions in aqueous solution. Mostly calcium ions have been employed for this purpose (Williams 2020).

Scheme 1. Two mechanisms for pectin chain degradation occurring at low (top) and high (bottom) pH values. At low pH values the glycosidic bond is cleaved, at high pH values a -elimination occurs preferably at repeating units bearing a methyl ester moiety.

Additional groups found in naturally occurring pectin are ferulic and acetic acid. They have a negative effect on the water solubility of the PS and the ability to form gels with metal ions (Kouwijzer et al. 1996). A chemical modification of pectin frequently occurring as a side reaction is the ester cleavage in basic or acidic aqueous systems. In these procedures also the ferulic- and acetic acid moieties bound to the pectin are cleaved, leaving a LMP. These procedures have to be considered critically, due to the pH sensitivity of the pectin chain, which leads to depolymerization (Albersheim et al. 1960). In the case of acidic treatment, the glycosidic bond can be cleaved (Scheme 1, top). In basic medium, a -elimination leads to fission of polymer chains as well (Scheme 1, bottom).

Although a review dealing with modification of pectin was published in 2015, which describes, besides chemical, also physical transformations (degradation) of the PS (Chen et al. 2015), it was reasonable to review the state of the art in this field due to the increasing popularity of PS as feedstock for sustainable materials. The present review is focused on chemical transformations of pectin and the products obtained, considering preferably the last 10 years of pectin research including correct chemical terminology. A common term used in PS chemistry is the degree of substitution (DS). The DS describes the (average) number of substituent groups attached per repeating unit of the PS. For cellulose, the value ranges from DS = 0 to 3, whereas for pectin from DS = 0 to 2 (EPST 2011). The amount of chemical modification at the carboxylic acid function of pectin is commonly expressed in percent conversion or degree of formation, not to be confused with the DS value. No degradation reactions have been specifically described. However, it can be assumed that -at least partially- pectin depolymerization occurs in the course of most of the reactions mentioned here. The review is divided into chemical reactions considering the carboxylic acid function at the C6 position and reactions considering the hydroxyl groups of pectin. In the last part, reactions not fitting in these categories are presented. Additionally, some trends concerning pectin and its applications are presented as well.

Formation of Esters at C6

The ester cleavage of the galacturonic acid unit of pectin is a side reaction occurring in extraction of pectin (Krall and McFeeters 1998; Diaz et al. 2007). The content and the type of ester makes it possible to modify the water solubility, viscosity, and gelling behavior of pectin (Flutto 2003). Besides the introduction of a hydrophobic group, which lowers the water solubility, additional functional groups can be introduced as well. Recent methods involve working with alkylation agents and employing the carboxylate form of pectin to obtain ester functions. The carboxylate reacts as a nucleophile with the alkyl halide. Zheng et al. (2013) employed a heterogeneous approach, comparable with the conditions of carboxymethylation of cellulose (Heinze et al. 2018). A highly esterified pectin was dispersed in 2-propanol and an excess of aqueous NaOH added (Scheme 2).

Scheme 2. Esterification of pectin in basic medium with long chain alkyl bromides at elevated temperatures in the presence of tetrabutylammonium (TBA) bromide as catalyst

To the mixture, the catalyst tetrabutylammonium (TBA) bromide and long chain alkyl bromides were added. The reaction proceeded at 80 °C, yielding low amounts of carboxylic acid ester only. The content of carboxylic acid moieties esterified ranged from 4% up to 14%. The water solubility decreases with increasing amount of ester formed, due to the introduction of these hydrophobic alky groups. In this procedure, TBA bromide was found to be a good phase transfer catalyst, which leads to the formation of hydrophobic pectin derivatives. Investigations showed that in the absence of the catalyst or a change of the counter ion of the ammonium salt to hydroxide, the reaction did not produce the desired hydrophobic pectin.

To perform a homogeneous alkylation of pectin in dimethyl sulfoxide (DMSO), the solubility of pectin had to be adjusted. Usually salts of pectin (alkali ions) are insoluble in DMSO. However, the TBA salt of pectin can be dissolved in this dipolar aprotic solvent (Renard and Jarvis 1999). By neutralizing pectin in aqueous solution with TBA hydroxide and subsequent lyophilization, a DMSO-soluble pectin salt can be obtained. The salt dissolved in DMSO was reacted with long chain alkyl bromides with C6, C12, and C18 alkyl chains (Liang et al. 2015). The alkyl ester formation with the alkyl bromides (C6) reached 0.7%, 2.4%, and 3.6%, respectively. To compare pectin with longer alkyl chain ester moieties as well, derivatives with C12 and C18 have been prepared from their corresponding alkyl bromides as well. The esterified compounds possessed a degrees of esterification of 0.7%. Interestingly the molecular masses were comparable after synthesis, as has been shown by high performance size exclusion chromatography measurements. The intrinsic viscosity [] was found to decrease with increasing alkyl chain length. On the contrary, their apparent viscosities increased with increasing length of the alkyl chain attached. The emulsion stabilizing effects of the esters obtained have also been probed and were shown to be correlated with the alkyl chain length of the ester moiety. The longer the alkyl chain attached to the pectin, the smaller the emulsified droplets turned out to be. Additionally, an increase in alkyl chain length of the pectin ester increased the stability of the emulsions formed, which was probed for a period of 7 days.

An enzymatic esterification of the carboxylic acid function of pectin could be achieved when employing laccase as an enzyme catalyst (Karaki et al. 2017). The reaction was performed in phosphate buffer at 30 °C, leading to an esterification of the hydroxyl group of ferulic acid with the C6 carboxylic function of pectin with a conversion of 6%. The low degree of esterification was difficult to detect with infrared (IR) and 13C nuclear magnetic resonance (NMR) spectroscopy. However, UV/Vis spectroscopy could show differences between the native and transformed pectin, due to the increase of aromatic moieties in the latter (Scheme 3). The study shows that the preparation of pectin derivatives with potentially antioxidant properties is possible, e.g., for the use in the food industry.

Scheme 3. Enzymatic esterification of pectin with ferulic acid in an aqueous buffer/tetrahydrofuran (THF) mixture at 30 °C

Along these lines, other phenolic acid derivatives closely related to ferulic acid have been enzymatically esterified with pectin as well (Zhang et al. 2020a). Gallic acid was bound via its 4-OH group with the C6 carboxylate of pectin employing a lipase (Novozyme 435, from Aspergillus niger) in a water/tetrahydrofuran mixture at 50 °C. Subsequently p/m/o-hydroxybenzoic acid and 3,4-dihydroxy benzoic acid have been reacted in a similar manner (Scheme 4, Zhang et al. 2020b). The materials obtained have been tested for their emulsifying properties as well as their antioxidant and antibacterial activities.

Scheme 4. Enzymatic esterification of pectin with different hydroxybenzoic acid derivatives in a water/tetrahydrofuran (THF) mixture at 50 °C

Fig. 2. Emulsifying properties of the starting pectin (a) and the pectin benzoic acid ester derivatives (b, c). Reprinted from International Journal of Biological Macromolecules, 165, Zhang, G., Zheng, C., Huang, B. and Fei, P., “Preparation of acylated pectin with gallic acid through enzymatic method and their emulsifying properties, antioxidation activities and antibacterial activities”, p. 202, Copyright (2020) with permission from Elsevier

The materials esterified showed an increase in emulsifying properties, e.g., decreased droplet size and prolonged droplet stability in contrast to the staring pectin, due to the amphiphilic nature of the hydrophobic aromatic carboxylic acid moiety introduced (Fig. 2). Moreover, they exhibited a clear inhibition zone in contrast to the native pectin when probing their antibacterial properties against E. coli and S. aureus. Interestingly in these investigations, no cross-linking or esterification of the benzoic acid derivatives with the hydroxyl groups of pectin was observed.

An esterification of the carboxylic acid group of pectin was also achieved by employing a photoactive diazo-resin (Plewa et al. 2011). The material, synthesized in a condensation reaction of diphenylamine-4-diazonium salt and paraformaldehyde, can generate phenyl cations under UV light irradiation. These cations are prone to nucleophilic attack from a carboxylate group, forming phenyl esters. By this photochemical reaction, pectin and the diazo resin could be cross-linked permanently. The reaction was monitored and quantified directly employing UV/Vis spectroscopy. The two polymers have been deposited in multiple layers on quartz plates and onto silica gel particles as well for the transformation and were subsequently cross-linked with UV light at 350 nm (Scheme 5). The resulting materials were shown to be promising scaffolds for growing cell cultures especially for bone tissue engineering.

Scheme 5. Formation of pectin ester followed by cross-linking of pectin and a diazo resin triggered by irradiation with UV light at 350 nm

Heteroatom Transformation at the Carboxylic Acid Group

Formation of amide

Pectin amide is a well-known thickening agent that is safe for use in the food industry (Mortensen et al. 2017). It can be produced simply by reacting native pectin with ammonia, thus cleaving the methyl ester moieties releasing methanol (Einhorn-Stoll et al. 2001). Structurally more diverse amides of pectin have been synthesized in the last few years.

An increased functionality of pectin derivatives can be achieved by reacting HMP with amino acids. The amide structures formed are stable chemical bonds, and the other functionalities of the amino acid moiety are still accessible. Kurita et al. (2012) reported on this transformation; HM citrus pectin and glycine, or glycine methyl ester, and glycylglycine were reacted in aqueous solution at pH value of 5.4 (Scheme 6). The amide formation occurred to an extent of 1% to 5% only, not changing the solubility of the biopolymer derivatives in water to a large extent. It is reasonable to assume that the water employed caused the low degree of amide formation due to hydrolysis of the pectin methyl ester in the acidic medium.

Scheme 6. Formation of pectin amides from high-methoxy pectin and glycine derivatives in water

The amide formation was also studied with cysteine and a LMP, thus incorporating thiol functional groups in the pectin structure (Chen et al. 2018; Eliyahu et al. 2021). The carboxylic acid function of pectin was activated in water using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The activated PS was reacted with L-cysteine monohydrate hydrochloride at a pH value of 4.75 to obtain the corresponding pectin amide (Scheme 7). The product fabricated was mixed with a chitosan acrylate, and the gel formation was studied in detail. Two cross-linking mechanisms could be identified. An electrostatic cross-linking was achieved, combining the carboxylate groups of pectin with the ammonium groups of the chitosan. A Michael addition of the thiol groups (Michael donor) to the electron-deficient double bond of the acrylamide moieties (Michael acceptor) resulted in a slow increasing permanent cross-linking of the two polymers mixed.

Scheme 7. The carboxylic acid function of pectin was activated in water with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and reacted with cysteine to form a pectin amide (top). A Michael addition between the thiol group of the pectin derivate and a chitosan acrylate resulting in a cross-linked gel (bottom)

Fig. 3. Anti-adhesive films based on disulfide cross-linked cysteine moieties bearing polygalacturonic acid amide. The material is shown in the dry state (A), immersed in phosphate buffered saline solution (B), and as a comparison a hyaluronate/carboxymethylcellulose gel (C). Reprinted from J. Mater. Sci.: Materials in Medicine, 24(6), Peng, H.-H., Chen, Y.-M., Lee, C.-I. and Lee, M.-W., “Synthesis of a disulfide cross-linked polygalacturonic acid hydrogel for biomedical applications”, p. 1378, Copyright (2013), with permission from Springer Nature.

The influence of temperature and pH value on the curing time and gel properties was investigated, showing that the increase of pH value gave stiffer gel structures and pH values below the pKa of chitosan led to materials with greatly enhanced adhesiveness. The gels are potential candidates for mucosa-mimetic materials (Fig. 3).

An amide-linked polygalacturonic acid-cysteine material was prepared with a degree of modification of 16%, which forms hydrogels via disulfide cross-linking (Peng et al. 2013). The hydrogel obtained could be employed as a carrier for rosmarinic acid and used to prevent postsurgical adhesion and inflammatory reactions. It was shown to significantly reduce adhesion incidents (over 90%); on the contrary hyaluronate/ carboxymethylcellulose leads to a reduction of 42% only.

To protect the thiol group against oxidation at pH values above 5, a cysteine-nicotinic acid disulfide was synthesized (Hintzen et al. 2013). This product was allowed to react with pectin after activation of the C6 carboxylic group with EDC in water. In addition to the EDC activation of the carboxylic acid, N-hydroxysuccinimide was added, probably to form an activated ester in situ right before the amide formation (Scheme 8). The resulting materials were shown to be non-cytotoxic on Caco-2 cells within 24 h. The S-protection improved the stability as well as the adhesive and cohesive properties of the pectin derivative compared to the unprotected pectin derivative. Comparable products were investigated for a drug release system, employing lidocaine as a model drug (Hauptstein et al. 2013). The gel formation of the starting pectin, pectin cysteine amide, and thiol protected pectin-cysteine amide were compared, and the swelling behavior and drug release of these gels in a saline buffer solution was probed for possible application in the buccal tissue. The thiol protected pectin-cysteine amide showed the best drug release behavior in comparison to the other two biopolymer derivatives investigated.

Scheme 8. Pectin amide formation with S-protected cysteine in water aided by activation of the carboxylic acid function by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)

In a more recent study, amino acids (glutamic acid, glycine, cysteine, lysine, and arginine) were allowed to react with HMP without any catalyst in water at 40 °C (Chen et al. 2020). From the amino acids tested, only glycine, lysine, and arginine could form amide bonds with the pectin. The degree of amide formation was generally low, not exceeding 6.5%. The pectin derivatives were probed using elemental analysis, Fourier transform infrared (FTIR), and 1H NMR spectroscopy. The surface topographies of the samples prepared showed appreciable differences as revealed by environmental scanning electron microscopy (SEM) (Fig. 4). The amino acid-pectin amide derivatives possess no cytotoxic effects, which would be expected considering pectin and amino acids are already non-cytotoxic.

The formation of a carboxylic acid amide with taurine led to a product that exhibits good surfactant properties (Aris et al. 2017). Taurine can be linked to polygalacturonic acid (PGA) via activation with EDC or without activation by treating the reaction mixture in a microwave oven (Scheme 9). The amount of carboxylic acid amide formed corresponding to the available carboxylic acid functions was 10%. The product exhibited surface activity comparable to Triton X-100 and, additionally, it is non-toxic against human dermal fibroblast and human leukemic cell lines.

Fig. 4. Environmental scanning electron microscopy images of the starting pectin, the pectin amide of glycine (P-Gly), the lysine (P-Lys) and the arginine derivative (P-Arg) of pectin after freeze-drying. Reprinted from Journal of Food Chemistry, 309, Chen, J., Niu, X., Dai, T., Hua, H., Feng, S., Liu, C., “Amino acid-amidated pectin: Preparation and characterization,” p. 5, Copyright (2020), with permission from Elsevier

Scheme 9. Formation of pectin amide of taurine in water, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC)

The synthesis route employing LMP with activation in aqueous medium via EDC was also followed by Tang et al. (2010). They used this method to form pectin amide with adriamycin, a drug employed in treating different types of solid malignant tumors. After dissolving all components, the reaction proceeded at 50 °C for 8 h. The dialyzed and lyophilized product was transformed into nanoparticles (NP) that showed a narrow distribution with the main peak at 152 nm and a Z-average diameter of 126 nm, which is a good value for the necessary renal clearance after NP administration. The drug release at 37 °C was increased by decreasing the pH-value from 7.4 to 5.0. The addition of lysosomes to the pectin derivative solution also had a positive drug releasing effect. Unfortunately, the study revealed that at concentrations equivalent to 0.125 to 1.000 µg of pectin derivative/mL, it did not inhibit the growth of either A594 or B16 cells to the same extent as the free drug or a mixture of the free drug with pectin. However, an anticancer effect on C57BL/6 mice was superior for the drug loaded pectin derivative in contrast to the drug alone or a mixture of the drug and pure pectin.

Using a water/dioxane mixture and EDC as activating agent, Perera et al. (2010) were able to form amide bonds between the carboxylic acid function of pectin and 4-aminothiophenol. The idea was to employ the thiophenol moiety as a reversible cross-linker in an oxidation/reduction procedure. Two derivatives were synthesized with a degree of amidation of approximately 3% and 10%. The viscosity of the sample dissolved was measured before and after the addition of hydrogen peroxide. It was found that a large increase in viscosity appeared. For the pectin derivatives containing 3% and 10% of amide groups, a 29-fold and 500-fold increase in dynamic viscosity was found, after 60 min of peroxide treatment. The materials synthesized showed increased water uptake after cross-linking. Additionally, no increase in toxicity compared to the starting PS was observed. Due to the increased PS stability after cross-linking, these pectin derivatives might be of interest for colon drug delivery systems.

Formation of hydroxamic acid

The reaction of hydroxylamine and HMP conducted in water at room temperature (RT) for 4, 18, 24, or 48 h resulted in the formation of a hydroxamic acid (Bae et al. 2011). The content of hydroxamic acid ranged from 4.7% to 10.4% conversion and increased with increasing reaction time (Scheme 10). In contrast to the highly esterified starting material, the hydroxamic acid derivatives produced showed an increased water solubility, which can be attributed to the decrease in hydrophobic methyl ester moieties. The inhibitory activity of pectin hydroxamic acid derivatives against semicarbazide-sensitive amine oxidase and angiotensin-converting enzyme was shown earlier (Hou et al. 2003).

Scheme 10. Formation of a hydroxamic acid derivative of pectin by reaction of a highly methyl esterified pectin in water with hydroxylamine

 

Formation of hydrazide

A homogeneous synthesis of pectin hydrazide was presented by Guo et al. (2014). The transformation of HMP was studied in aqueous solution with a large excess (60 eq.) of hydrazide hydrate at 80 °C for 12 h (Scheme 11). The hydrazine hydrate reacted with the methyl ester groups of pectin, forming the corresponding hydrazide in only moderate yields. The product showed an increased nitrogen content and was able to bind mercury ions from an aqueous solution more efficiently than the starting HMP.

Scheme 11. Homogeneous reaction of high-methoxy pectin with hydrazine hydrate in water at elevated temperatures

A heterogeneous synthesis approach for pectin hydrazide could be realized as well and was shown to be more efficient and even useful for industrial application (Würfel et al. 2021). The reaction of LMP or PGA proceeds in a slurry of 2-propanol between RT and 50 °C, applying 4 eq. to 10 eq. of hydrazide hydrate. The reaction is complete after 4 h, leading to a hydrazide content of up to 98%. Interestingly, no methyl ester groups are present or needed for this transformation. The carboxylic acid groups or their ammonium salts react readily with hydrazine hydrate while releasing water or ammonia by forming the correspondent hydrazide (Scheme 12). The pectin hydrazide thus obtained had a good metal chelating ability and can form stable gels with multivalent metal ions. Furthermore, an ex ovo hen’s egg test demonstrated that the pectin hydrazide derivatives are non-toxic.

Scheme 12. Formation of polygalacturonic acid hydrazide from ammonium polygalacturonate heterogeneously in 2-propanol

Acylation Reaction at the Hydroxyl Group

Native pectin incorporates varying amounts of acyl groups, originating from acetic- and ferulic acid groups. The acetylation of pectin was investigated and was shown to have a strong influence on the water solubility and the ability to form gels with multivalent metal ions (Wang et al. 2019). Different strategies have been employed to acylate pectin with a variety of carboxylic acid derivatives. A solvent-free approach was published for esterifying the hydroxyl groups of pectin with fatty acid anhydrides catalyzed with base at 160 °C (Monfregola et al. 2011). The compounds were ground in an agate mortar and heated in an oil bath for 15 to 20 min (Scheme 13). The resulting pectin esters have been isolated by extracting the reaction product with chloroform. This method of purification contradicts the claim of an environmentally friendly method of esterification, as stated. The reaction yields pectin esters of low DS of 0.27 for hydroxyl oleic acid anhydride and DS = 0.07 for linoleic acid anhydride.

Scheme 13. Solvent-free and base-catalyzed acylation of pectin with long chain acid anhydrides at 160 °C for 15 min

Later, a similar microwave assisted approach was published (Calce et al. 2012). The mixture of pectin, fatty acid anhydride, and K2CO3 was ground and treated subsequently in the microwave with 900 W for 3 min to 6 min. The products were still water soluble and possessed an intense IR absorption belonging to the C-H stretching vibrations of the fatty acid moieties introduced. The anhydrides of linoleic acid, oleic acid, and palmitic acid were employed. As interesting as this synthesis protocol seems to be, the reactions was performed in amounts of 30 mg only, showing problems arising from solvent-free procedures such as homogeneous mixing of the compounds employed and efficient absorption of the microwave irradiation by materials with low dielectric properties.

Pectin derivatives incorporating linoleic-, oleic-, and palmitic acid esters were found to be active against different strains of bacteria (Calce et al. 2014). Pectin oleate and -linoleate could inhibit the growth of S. aureus and E. coli by 50% to 70%. Furthermore, the pectin esters could be coated on polyethylene films that possess good oxygen barrier properties, which could be interesting in the field of food application and packaging.

The reaction of HMP with diacylhalides (glutaryl chloride and sebacoyl chloride) was described (Seslija et al. 2018). The homogeneous transformation with the acylation reagent proceeds in DMSO and pyridine applying a ratio of reagent/pectin of 1:3 and 1:15 (Scheme 14). The conversion rate was low. Otherwise the resulting product would have been insoluble in water, due to cross-linking reactions. The inefficient reaction and the use of DMSO as solvent raises the question, if this synthesis protocol is really suited to produce hydrophobic pectin derivatives for food packaging films as proposed by the authors.

Scheme 14. Homogeneous acylation of pectin with diacyl halides in dimethyl sulfoxide (DMSO) at 50 °C for 12 h

Expanding the functionality of pectin further, the PS was modified with thiol group bearing compounds, including thioglycolic acid (Sharma and Ahuja 2011). The esterification proceeded with an excess of thioglycolic acid in water aided by HCl as catalyst (Scheme 15). A biopolymer derivative with a thiol content of 0.6 mmol/g was obtained. This value can be approximated to a DS of 0.1. Neither elemental analysis nor NMR data are available for the product. The thiol groups have been quantified with an Ellman’s test and the ester formation was analyzed by IR spectroscopy. The materials produced are still capable to form gels with Ca2+ and show an increased mucoadhesion time compared to the starting material.

Scheme 15. Acylation of pectin with thioglycolic acid in acidic aqueous medium at 80 °C

The introduction of unsaturated ester groups in the pectin structure was achieved by Almeida et al. (2015). The reaction employed pectin in N,N-dimethyl formamide that was reacted with maleic anhydride at 70 °C for 24 h without a catalyst. The esterified pectin obtained bears maleate groups with a DS of up to 0.24, as determined with 1H NMR spectroscopy. The esterification increased the thermal stability of the pectin derivative, which started substantial degradation at 268 °C. This temperature is 18 °C above the degradation temperature of the staring pectin. The material obtained showed an increased inhibition efficiency against Caco-2 colon cancer cells compared to the starting pectin. In addition, the cytotoxicity of the PS was also lowered by this modification.

Si-O Bond Formation

The formation of Si-O bonds is a reaction closely related to acylation. The bonds formed can be inert depending on the neighboring substituents at the silicon atom due to steric hindrance or easily cleavable, e.g., by acid-catalyzed hydrolysis. A condensation reaction of pectin with 3-aminopropyltriethoxysilan (APTES) in toluene was reported, resulting in an amino group bearing biopolymer derivative (Baran 2019). The resulting nucleophilic pectin derivative was further treated with thiophene-1-carbaldehyde to form a Schiff base. This imine structure was employed as a ligand for a palladium complex, which could be applied for efficient Suzuki-Miyaura reaction (Scheme 16). The catalyst could be used without activity loss for 12 catalytic cycles. The Pd-loaded pectin derivative (14 w% Pd) showed no leaching of the metal.

Scheme 16. Condensation of 3-aminopropyltriethoxysilan with pectin, imine formation with thiophene-1-carbaldehyde, and Pd complexation led to a polysaccharide derivative fit for Suzuki-Miyaura reaction (adapted from Baran 2019)

Very recently, a homogeneous approach to prepare silylated pectin derivatives was published employing PGA in dry formamide (FA) with trimethylsilylchloride (TMSCl)/imidazole or 1,1,1,3,3,3-hexamethyldisilazane (HMDS) as reagent (Würfel et al. 2019a). The reaction was shown to be more efficient employing HMDS, leading to a trimethylsilyl polygalacturonic acid derivative with a DS of 1.8, on the one hand. On the other hand, TMSCl was found to be much less efficient, leading to a DS of 0.60 only (Scheme 17). A complete silylation of the hydroxyl groups was not feasible even with a large excess of reagent (12 eq.). The silylated pectin derivative obtained was prone to hydrolysis in water. Additionally, side reactions could be identified, which led on the one hand to cross-linking with TMSCl, and on the other hand to the formation of amide groups with HMDS during the reaction.

Scheme 17. Silylation of polygalacturonic acid with trimethylsilyl chloride (TMSCl)/imidazole or 1,1,1,3,3,3-hexamethyldisilazane (HMDS) in formamide resulting in trimethylsilyl polygalacturonic acid derivatives (adapted from Würfel et al. 2019a)

S-O-bond Formation

The synthesis of pectin sulfate and their anticoagulant activity was studied (Vityazev et al. 2010). For the sulfation reaction of pectin, pyridine monomethyl sulfate, pyridine sulfur trioxide, and chlorosulfonic acid have been investigated (Scheme 18). All three sulfation reagents employed led to a partial PS degradation. The sulfating reagent with the best results considering degree of sulfation, degree of degradation, and product yield turned out to be the chlorosulfonic acid. The specific anticoagulant activity of the products obtained depends further on the plant species employed for pectin extraction, the composition of the pectin employed, and the degree of sulfation introduced. The specific antithrombin activity of the biopolymer derivatives obtained was assayed and showed to be superior in contrast to the starting pectin.