Hydrolyzed galactomannans

Hydrolysis, either by chemical methods or enzymatic reactions, is one of the most important and practical strategies for the modification of polysaccharides such as GM (^{Geronço et al., 2021}). In the enzymatic method, the galactose side chains or the mannose main chain, can be hydrolyzed by α-galactosidase and β-mannanase, respectively, it is simple to control, and the reaction conditions are mild compared to chemical hydrolysis which is non-specific and highly aggressive (^{Ba et al., 2012}). The GM functional properties are greatly influenced by their molecular weight, and based on this, the hydrolysis of the mannose main chain and galactose side chain results in a strategy for the modification of its viscoelastic properties (^{Zhou et al., 2016}).

For this purpose, hydrochloric, sulfuric, and trifluoroacetic acids are used, in addition to the application of heat to accelerate the hydrolysis reaction of the side chains by mild conditions (^{Biermann, 1989}). However, this type of hydrolysis results in a difficult elimination of acidic remnants with the probable exception of trifluoroacetic acid, which can be removed by rotary evaporation due to its volatility at not very high temperatures (^{Liu et al., 2020}). Hence, research on obtainment and fractionation of GM hydrolysates has focused mainly on the use of enzymatic catalysis as a less complicated way to obtain mannooligosaccharides.

^{Wu et al. (2019)} evaluated the hepatoprotective effect of GM partially hydrolyzed with β-mannanase from Rhizomucor miehei. The composition of these hydrolysates was 24.9 % (w/w) of mannooligosaccharides with a degree of polymerization of < 7. The average molecular weight of the hydrolyzed GM was 2.5 × 10⁴ Da identified by gel permeation chromatography, the decrease of which was effective by enzymatic treatment. These hydrolyzed GM had a hepatoprotective effect in Kunming mice, by increasing the activity of the superoxide dismutase and glutathione peroxidase antioxidant enzymes. This event, in turn, decreased alcohol-induced lipid peroxidation, and thus the integrity and fluidity of cell membranes.

Similarly, ^{Chen et al. (2018)} hydrolyzed GM with 8 U/mL of a Pichia pastoris thermostable β-mannanase at different times of action on the substrate at 50 °C. Native GM had a higher molecular weight than the hydrolyzed ones with a significant decrease from 5.58 × 10⁶ to 3.18 × 10³ Da, demonstrating the presence of mannooligosaccharides after 24 h. These hydrolysates exhibited immunomodulatory activity by stimulating the production of TNF-α in RAW 264.7 cells, an important cytokine that intervenes in the inflammation and apoptosis processes.

Recently, hemicellulosic polysaccharides such as GM and their degradation products are gaining more attention due to their excellent in vitro and in vivo antioxidant enhancement and immunomodulatory activities. ^{Tao et al. (2021)} hydrolyzed GM from β-endo-mannanase of Trichoderma reesei and evaluated its antioxidant and immunomodulatory effect. GM hydrolysates showed a strong protective effect against induced oxidative stress, using a model of RAW 264.7 cells injured with hydrogen peroxide, as indicated by superoxide dismutase activity and malondialdehyde content.

Structurally, mannooligosaccharides continue to be only the repeating unit of GM in its native form, as its general composition and most of its physicochemical properties are not affected. For example, the FTIR spectrum is very similar to GM and their hydrolysates that show characteristic absorption bands of this polysaccharide as reported in the literature (^{Cerqueira et al., 2011}; ^{Mudgil et al., 2012}). Furthermore, the partial hydrolysis of GM leads to a decrease in the polydispersity index by up to one-third, this is due to the general reduction of all molecular weight populations (^{Buriti et al., 2014}). Partially hydrolyzed GM, as it does not suffer significant alterations, seems to be a potential alternative, increasing the availability of new food ingredients, as a source of dietary fiber.

Sulfated galactomannans

A promising derivatized structure of GM is the synthesis of their compounds that contain a sulfonic group (-SO₃H) or their salt (-SO₃Na). The addition of this functional group in a biopolymer macromolecule changes its physicochemical characteristics, increasing its solubility in water and its biodegradability (^{Kazachenko et al., 2020}). The presence of the -SO₃H group in the polysaccharide structure (Figure 3) provides important properties, since the sulfate group has a negative charge in a determined pH range to electrostatically bind to positively charged molecules. However, the synthesis of sulfated polysaccharides is a challenge due to the many stereocenters in their structure, the presence of similar functional groups, and the need to preserve the orientation of the glycosidic bonds. Therefore, from a chemical perspective, sulfated polymeric structures are synthesized by sulfating natural polysaccharides or polymeric analogs (^{Caputo et al., 2019}).

Figure 3 Scheme of sulfation process of galactomannans (^{Muschin et al., 2016}). 

Figura 3: Esquema del proceso de sulfatación de galactomananos (^{Muschin et al., 2016}) 

There are many challenges associated with polysaccharide sulfation, such as low -OH group reactivity, poor solubility in organic solvents, regioselectivity, degree of sulfation, and isolation of the derivatized polysaccharide. There are scarce solvents in which both the polysaccharide and the sulfated derivatized are soluble, and the reactions are not complete (^{Richter and Klemm, 2003}). On the other hand, identifying and targeting a specific sulfation site is complicated due to the many -OH groups in the polysaccharide. Thus, protecting groups are required which increases the complexity, and decreases yield. Furthermore, sulfating multiple -OH on a single monosaccharide unit is complicated due to electrostatic repulsion between neighboring -SO₃H adding a grade of complexity to the derivatization (^{Al-Horani and Desai, 2010}).

Given the structure-function relationship of polysaccharides, the degree of sulfation becomes an important parameter to control, when synthesizing sulfated GM (^{Caputo et al., 2019}). The chlorosulfonic acid and sulfuric acid are currently used in the sulfation process of GM as a sulfating reagent in presence of a strong base (pyridine or piperidine), and its subsequent isolation by converting it into sodium salt. In this sense, the use of chlorosulfonic acid is the most used route to obtain sulfated GM with different sulfation degrees. In contrast to these reagents, sulfamic acid is a stable non-hygroscopic crystalline substance with the reactivity like that of the -SO₃ tertiary amine complex that in the presence of urea, which plays a role of the main catalyst, its sulfation occurs (^{Kuznetsov et al., 2018}).

In this sense, ^{Kazachenko et al. (2021)} synthesized sulfated GM from sulfamic acid-urea complexes at different reaction times and temperatures. In this study they determined that at 80 °C and for 60 min, the highest possible degree of sulfation (1.67) is obtained by this synthetic route. Thus, from these milder conditions it is possible to have a more effective sulfation process from eco-friendly reagents than from the conventional chlorosulfonic acid route. ^{Qin et al., (2020)} evaluated a response surface methodology (RSM) study for the optimization of the sulfation process by varying the chlorosulfonic acid / pyridine ratio, temperature, and reaction time. This study provided optimal sulfation conditions for GM with a 66 % degree of sulfation, which were an acid chlorosulfonic / pyridine ratio of 3:1, reaction time of 4 h and reaction temperature of 40 °C. Two characteristic absorption peaks appeared in FTIR spectra at 1250 cm^-1 and 806 cm^-1, indicating sulfation of GM. The ¹³C-CP/MAS NMR spectra confirmed that the substitution position was mainly at C6 in sulfated GM.

Previously, ^{Wang et al. (2014)} optimized the sulfation process by varying the same parameters by RSM. In this study, a mathematical model was constructed where the optimal reaction conditions of GM were a chlorosulfonic acid / pyridine ratio of 1.27, reaction time of 120 min, and reaction temperature of 50.8 °C. Among the three reaction parameters studied, reaction time was the most significant factor to have an effect on the sulfation process, based on the slope gradient in the 3D response surface plots (^{Wang et al., 2014}; ^{Qin et al., 2020}).

^{Lopes et al. (2013)} sulfated GM with chlorosulfonic acid in pyridine/formamide to study the inhibition of herpes simplex virus (HSV-1) and poliovirus (PV-1). These authors reported an 8.7 % degree of sulfation obtained from three consecutive sulfation procedures. FTIR analysis demonstrated the presence of sulfated polysaccharides with a band at 1255 cm^-1 which refers to the vibration of asymmetric stretching of the ester sulfate bond (S=O). These sulfated products inhibited HSV-1 and PV-1 with an IC₅₀ of 405 μg/mL and 1.73 μg/mL, respectively, interfering various stages of viral replication, adsorption, and penetration in both cases.

Similarly, ^{Mendes-Marques et al. (2015)} sulfated GM with the same method for the inhibition of dengue virus, in this case they reported a 33 - 39 % degree of sulfation and mentioned the importance of this parameter in its activity. Confirmation of the sulfation process was performed by FTIR spectroscopic analysis with the presence of a band at 1259 cm^-1 corresponding to the stretching vibrations of the S=O group mentioned above. Likewise, these derivatized polysaccharides presented a positive correlation between the degree of sulfation and the antiviral activity against the dengue virus DENV-2 and its antioxidant capacity.

Galactomannan fatty acid esters

Chemical modification has conventionally been used as a strategy to change the hydrophilicity of polysaccharides, through the formation of esters. The esterification of GM is carried out regioselectively, this is that the primary alcohol will always be more nucleophilic than the secondary alcohol in the polysaccharide structure (^{Kanelli and Topakas, 2017}). Their respective rate constants for the primary and secondary alcohols do not change during the esterification process, where primary alcohol reacts faster than secondary alcohol, decreasing its concentration as the reaction progresses. Therefore, eventually, the reaction rates will become similar and the regioselectivity will decrease (^{Fox et al., 2011}; ^{Koschella et al., 2006}).

Esterification generally involves the reaction of an alcohol with an acylating agent, such as vinyl carboxylates, acyl chlorides, acid anhydrides, as well as the carboxylic acids themselves. On this basis, carboxylic acids have been obtained by Fischer esterification conventionally by strong acid catalysis (concentrated H₂SO₄), or by in situ activation of the carboxylic group under mild conditions for the acylation of polysaccharides (^{Cumpstey, 2011}). Previously, different synthetic routes have been reported to obtain GM esters, through both chemical and enzymatic routes, either using strong acids or strong bases to react with the acyl donor or using enzymes such as lipase or transferases to catalyze the reaction.

Fujioka et al. (2009) synthesized GM esters through the esterification of the succinate group in the polymer chains. They reacted native GM with succinic anhydride, using DMSO as a solvent and 4-dimethylaminopyridine as a catalyst. FTIR spectroscopy was used to demonstrate the presence of the ester group, showing absent absorption bands at 1735 cm^-1 and 1550 cm^-1 in the spectrum of the native GM indicating the esterification of the polysaccharide with a degree of substitution of 0.6. In another study, ^{Shenoy and Melo (2010)} esterified hydroxypropyl GM using acetic anhydride as donor acyl and pyridine as a catalyst, to be used as filler in unsaturated polyester compounds. The esterification of this polysaccharide leads to an increase in the hydrophobic character of the resulting polymer where the variation of the reaction time results in the formation of esterified GM with degrees of substitution of 0.58, 1.1, 1.8, and 3.0. The FTIR of the products showed an increase in the intensity of the carbonyl band obtained in the region of 1735 - 1750 cm^-1 derived from the increase in the degree of substitution of the polymer.

On the other hand, ^{Iqbal et al. (2020a)} reported GM esters of aliphatic carboxylic acids of medium and long chain (C5-C18) from acyl chlorides, examining their physical properties concerning the length of the fatty acid chain and degree of substitution. The FTIR spectrum showed typical bands of a structurally native GM for all samples, added to the bands at 1754 cm^-1 and 723 cm^-1 corresponding to the C=O bond of the ester group and the oscillating vibrations of the -CH₂ groups of the aliphatic chain, respectively. However, this type of reaction leads to polymer degradation and not very high degrees of substitution due to the use of aggressive catalysts with respect to pH.

An alternative to this process has been biocatalysts through the use of enzymes to modify the structure of polysaccharides, with the advantage that it is often chemo specific, which allows the synthesis of products with well-defined or stereo-specific structures (^{Cumpstey, 2011}; ^{Cheng and Gu, 2012}). Some enzymes have a wide tolerance to the substrate and catalyze reactions in generic structures, hence, esterases, proteases, and lipase have been used to catalyze the formation of esters in polysaccharides, even being an unnatural function of the enzyme. This is because they do not differentiate the -OH groups, but the enzyme will tend to act on the -OH group (esterification) or ester (hydrolysis) that is more sterically accessible, that is, located in primary positions (^{Cumpstey, 2011}). Enzymes normally require at least an amount of water to carry out their action correctly, being structurally unstable in non-aqueous media. However, water is not a good medium for ester synthesis, in these conditions is reversible. When the water is in excess, the chemical equilibrium of the reaction tends towards hydrolysis, so the degree of substitution of the products would be very low (^{Alissandratos et al., 2010}).

On the other hand, polar solvents such as DMSO and DMF can remove essential catalytic water from the surface of enzymes, inactivating them and thus have lower hydrogen bonding capacity leading to higher enzymatic activity (^{Gu and Cheng, 2005}). However, there are no well-established protocols for GM enzymatic esterification, unlike starch and cellulose, which are the most widely used for this chemical modification (^{Yang and Wang, 2003}; ^{Rajan et al., 2008}). Most studies are mainly focused on their interaction with hydrophobic compounds and not on the application of GM esters.

In that context, ^{Sarkar and Singhal (2011)} elaborated GM succinate and oleate as microencapsulation material in O/W emulsions. The addition of these aliphatic chains leads to an improvement in its emulsifying capacity with low droplet size and viscosity, which makes it an important material for the protection of different food components. In another study, ^{Cerqueira et al. (2019)} elaborated GM acetate nanomicelles as carrier material for hydrophobic compounds such as curcumin. These modifications to the GM led to an encapsulation efficiency greater than 99 % and a ζ potential of -18.1 mV, indicating an effective charge of curcumin and repulsion between the nanomicelles to prevent them from agglomerating.

In contrast, in the esterification of GM for the addition of aliphatic chains, hydrophobic films of GM acetate have also been developed as a barrier system and protection against water in food packaging (^{Liu et al., 2021}). In this work, the chemical modification provides a good hydrophobicity to the film with a contact angle of 107 ° and a decrease in its water vapor permeability properties. Thus, the synthesis of these amphiphilic compounds contributes to the development of new compounds for hydrophobic systems. In addition, it will be of vital importance when establishing the synthetic routes to produce GM esters and choose the appropriate compounds for the correct esterification of the polysaccharide.

Carboxymethyl galactomannan

Carboxymethyl galactomannan (CMG) is an anionic GM derivative with a high dissolving rate of viscosity and good stability. Various synthetic routes have been developed to obtain CMG (Figure 4). This is produced by reacting monochloroacetic acid or its sodium salt with deprotonated GM. A strong base, such as sodium hydroxide, is needed to activate the -OH group of GM for nucleophilic substitution (^{Cumpstey, 2011}). Previous studies related to CMG preparation used organic solvents such as acetone, ethanol, isopropanol, and butanol as the medium (^{Gao and Grady, 2018}). GM has been previously dissolved in water, however, the use of excess water in a ratio of approximately 50:1 generally has poor compatibility, because water can hydrolyze the alkylating agent (^{Dodi et al., 2011}).

Figure 4 Synthesis of carboxymethyl galactomannan (CMG) of guar gum (^{Iqbal et al., 2020}). 

Figura 4: Síntesis de carboximetil galactomanano (CMG) de goma guar (^{Iqbal et al., 2020}) 

The synthesis of CMG is widely studied; ^{Wang et al. (2021)} obtained CMG as robust water-soluble binder for silicon anodes in lithium-ion batteries, and the ¹H NMR spectrum shows that carboxymethyl was attached to position C-6. Additionally, in the FTIR spectrum, two new bands appear at 1602 cm^-1 and 1411 cm^-1 corresponding to the asymmetric and symmetric stretching vibrations of the COO- group respectively, which further confirms the carboxymethylation of GM. In another study, ^{Iqbal et al. (2020b)} synthesized carboxymethylated GM derivatives and their structural characteristics as a synthetic thickener for textile prints. These had a degree of substitution of 0.35, and the FTIR spectrum demonstrates the substitution of carboxymethyl in GM with the bands corresponding to the COO- vibrations that are symmetric or asymmetric at points 1429 cm^-1 and 1615 cm^-1, respectively.

The potential applications for CMG can be diverse, for example, ^{Verma et al. (2020)} evaluated the CMG pharmaceutical applications by making ionotropically gelled beads using sodium diclofenac as a model drug, optimizing its design by RSM. The formulation used was 2.85 % (w/v) CMG and 15 % (w/v) CaCl₂ where the beads showed a loading efficiency of 95.41 % and a release of 93.32 % of diclofenac during 24 h. In another study, ^{Li et al. (2021)} fabricated polyelectrolytic gels of CMG and quaternary ammonium GM for the removal of dyes in water contaminated with Congo red and methylene blue as study models. These gels were pH and ion sensitive, with maximum adsorption capacities of 1441 and 94.52 mg/g of Congo red and methylene blue, respectively, mediated by electrostatic interactions.

Galactomannan alkyl ethers

Etherification of polysaccharides involves the reaction of the saccharide alcohol groups with an alkylating agent, these include alkyl halides (e.g. bromides, chlorides, and iodides), or less commonly, alkyl sulfonates. Typically, a strong base is used to deprotonate a saccharide alcohol to form an alkoxide ion that attacks the alkyl halide, through an SN₂ mechanism to form a polysaccharide alkyl ether. Usually, the alcohol from which the alkoxide is derived can be used as solvent, but it improves the reaction performance if we use aprotic solvents such as DMF or DMSO (^{Cumpstey, 2011}). Currently, derivatized GM alkyl ethers have been reported, such as: methyl, butyl, benzyl, ethyl acrylate and 2-hydroxypropyl tri-methylammonium chloride (HPTAC) GM ethers (^{Kazachenko et al., 2022a}).

The synthesis of methyl and benzyl GM ether has been widely studied, for instance, ^{Mohammed and Ragab (2021)} synthesized these GM ethers in a DMSO/paraformaldehyde system using dimethyl sulfate and benzyl chloride as alkylating agents in presence of sodium hydroxide as a catalyst. The correct etherification of the polysaccharide was determined from FTIR shown new peaks at 1114 cm^-1 and 1056 cm^-1 indicating C-O stretching vibration of functional group of ether for methyl GM and benzyl GM respectively. In another study, ^{Kazachenko et al. (2022b)} synthesized GM butyl ether using bromobutane as an alkylating agent in the presence of sodium hydroxide as a catalyst. Etherification was confirmed by FTIR showing absorption bands at 1077, 879, 815 and 796 cm^-1, corresponding to the vibrations of the ether bond of the -C-O-C- groups.

The potential applications of GM alkyl ethers have not yet been as extensively studied. The etherification of HPTAC has been used as a strategy for obtaining cationic GM as a corrosion inhibitor (^{Biswas et al., 2017}). Similarly, ^{Singh et al. (2021)} synthesized galactomannan ethyl acrylate ether as a corrosion inhibitor on steel under acidic conditions. However, it is necessary to address the possible applications of all types of GM alkyl ethers including those synthesized from uncharged alkyl groups to obtain a more diverse picture in the use of this type of derivatized GM.

Phosphorylated galactomannan

The production of phosphorylated GM is carried out by different synthetic routes: phosphoric acid, phosphoric anhydride, phosphorous trichloride (Figure 5), or dimethyl phosphate (^{Suflet et al., 2006}). Furthermore, H₃PO₄ and H₃PO₃ have been usually used as phosphorylating reagents at higher temperatures, generally leading to a low degree of substitution (0.26, phosphate content 4.05 %) and polysaccharide degradation (^{Wang et al., 2013}). The applications for phosphorylated GM have not been so widely studied, being its antioxidant capacity and its relationship with DS one of the few reports found. In this sense, ^{Wang et al. (2014)} synthesized phosphorylated GM with POCl₃ to evaluate the effect of the degree of substitution with respect to its conformation in solution. The degree of substitution varied from 0.35 to 0.52, and the FTIR spectrum showed new peaks around 1250 cm^-1 that describe an asymmetric P=O stretching vibration, a shoulder at 1050 cm^-1 corresponding to the P-OH bond, which is attributed to the presence of phosphorylated GM. Finally, the antioxidant capacity was increased to a higher DS of 0.52, which indicates that this derivatized polysaccharide could be effective in scavenging radicals in vitro.

Figure 5 Schematic route for obtaining phosphorylated GM by POCl₃ (^{Wang et al., 2014}). 

Figura 5: Ruta esquemática para obtener galactomananos fosforilados mediante POCl₃ (^{Wang et al., 2014}).