A new study of iodine complexes of oxidized gum arabic: An interaction between iodine monochloride and aldehyde groups

Accepted Manuscript Title: A NEW STUDY OF IODINE COMPLEXES OF OXIDIZED GUM ARABIC: AN INTERACTION BETWEEN IODINE MONOCHLORIDE AND ALDEHYDE GROUPS Authors: Akbar Ali, Showkat Ali Ganie, Nasreen Mazumdar PII: DOI: Reference:

S0144-8617(17)31151-7 https://doi.org/10.1016/j.carbpol.2017.10.005 CARP 12854

To appear in: Received date: Revised date: Accepted date:

1-3-2017 8-9-2017 2-10-2017

Please cite this article as: Ali, Akbar., Ganie, Showkat Ali., & Mazumdar, Nasreen., A NEW STUDY OF IODINE COMPLEXES OF OXIDIZED GUM ARABIC: AN INTERACTION BETWEEN IODINE MONOCHLORIDE AND ALDEHYDE GROUPS.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A NEW STUDY OF IODINE COMPLEXES OF OXIDIZED GUM ARABIC: AN INTERACTION BETWEEN IODINE MONOCHLORIDE AND ALDEHYDE GROUPS Akbar Ali, Showkat Ali Ganie and Nasreen Mazumdar* Material (Polymer) Research Laboratory, Department of Chemistry, Jamia Millia Islamia Central University, New Delhi 110025, India Highlights    

Gum arabic was oxidized with periodate to generate dialdehyde groups in the chains The aldehyde groups in oxidized GA reacted with ICl and I2 forming iodine complexes The experimental and spectral data showed addition of ICl molecules to –CHO groups ICl treated oxidized gum released iodide, the nutritional form of iodine, in H2O

ABSTRACT

Gum arabic, a plant polysaccharide was oxidized with periodate to produce aldehyde groups by the cleavage of diols present in the sugar units. The oxidized gum was then iodinated with iodine monochloride (ICl) and the interaction between electrophilic iodine, I+ and reactive carbonyl groups of the modified gum was studied. Results of titrimetric estimation performed 1

to determine the extent of oxidation and aldehyde content in the oxidized gum showed that degree of oxidation ranged between 19.68—50.19% which was observed to increase with periodate concentration; the corresponding aldehyde content was calculated to be 5.15— 40.42 %. Different strengths of ICl were used to iodinate the oxidized gum and the iodine content of the complexes varied from 6.11—11.72 % as determined by iodometric titration. Structure elucidation of the iodine complexes conclusively established the attachment of ICl molecules to -CHO groups. A reaction scheme has been proposed suggesting an electrophilic addition of the reagent to the aldehyde groups, a mechanism that was also supported by iodide ion release studies. Keywords: Gum arabic; Oxidized gum arabic; Iodine-gum arabic complexes; Iodide ions; Iodine monochloride *Corresponding author; e-mail: [email protected]; Tel: +91-011-26981717 ext. 3259 1. Introduction Structurally natural gums are a mixture of polysaccharides that are hydrophilic in nature and produce simple sugars such as arabinose, galactose, mannose, rhamnose and glucuronic acid on hydrolysis; the other structural constituents of plant gums include proteins, Ca and Mg (Mirhosseini & Amid, 2012). The natural gums are used as important ingredients in pharmaceutical formulations and readymade food preparations. These can also be modified chemically to improve their properties and functionalities and widen the scope of their applications in other fields e.g. drug delivery systems, medicine etc. (Rana, Rai, Tiwary, Singh, Kennedy & Knill, 2011). Gum arabic (GA), a complex and branched polysaccharide is mainly obtained from Acacia senegal and Acacia seyal trees; the other sources include the related species of Acacia such as

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Acacia karroo, Acacia polyacantha, Acacia sieberiana etc. (Masuelli, 2013). The carbohydrate content of this water soluble biopolymer is ~ 97 % which is mostly composed of D-galactose and D-arabinose units; the protein component in GA present as arabinogalactanprotein and glycoprotein constitutes <3 % of the total gum (Mariana et al., 2012; Islam, Phillips, Sljivo, Snowden & Williams, 1997). This plant polysaccharide has been extensively used in its pure form as an emulsifier and thickener in food; in addition, its modified forms are also reported to be used as hydrocolloids in a variety of applications (Jeurissen, DiNovi & Larsen, 2010). Chemical modification of GA and application of its phthalate and succinate esters were reported (Ribero, Laurentino, Alves, Bastos, Costa, Canuto & Furtado, 2015) as early as in 1940 and the process of functionalizing this useful gum continues till today. Some of the recent reports include chemical modification of GA with sodium trimetaphosphate crosslinker for encapsulation of essential oils (Aguilar et al., 2010), octenyl succinic anhydride modified GA for use as a food additive (Sarkar & Singhal, 2011) and acetylation of GA for preparing iodine complexes (Ahmad, Mazumdar & Kumar, 2013; Ganie, Ali & Mazumdar, 2015). Some publications are available on periodate oxidation of GA that introduces aldehyde groups in the gum by the cleavage of vicinal diol structures and the usage of these aldehyde groups for covalently bonding drug molecules containing appropriate functional groups. Nishi and co-workers (Nishi & Jayakrishnan, 2004; Nishi & Jayakrishnan, 2007; Nishi, Antony & Jayakrishnan, 2007) used oxidized GA as a carrier for primaquine, an anti-malarial drug and ampicillin antibiotics; Stefanovic et. al. (Stefanovic, Jakovljevic, Gojgic-Cvijovic, Lazic & Vrvic, 2013) coupled oxidized GA with an antibiotic ‘nystatin’ through reaction between amine groups of the antibiotic and aldehyde groups of oxidized gum. GA-dialdehyde was also used as a crosslinker for gelatin to prepare gel scaffolds for cell culture (Sarika, Cinthya, Jayakrishnan, Anilkumar & James, 2014). Though gumaldehyde derivatives have been mostly used for preparing polymer-drug conjugates through

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Schiff reactions, no study has been reported on the reaction between GA-aldehyde and iodine monochloride (ICl) to introduce iodine into the polysaccharide. Natural polymer-iodine complexes are important compounds in the biological field (Yu & Atalla, 2005) and have been obtained by treating xylan (Abe, 1958), cellulose (Takahashi, 1987), chitin (Yajima et al., 2001), chitosan (Takahashi, 1984), alginate (Lecker, Kumari & Khan, 1997) and glycogen (Moulay, 2013) using molecular iodine vapour and/or an aqueous solution of I2/KI. Recently, our group prepared iodine complexes of GA derivatives with ICl and studied the interaction between electrophilic iodine (I+Cl-) and polar carbonyl groups introduced in the natural polymer through acylation (Ganie, Ali & Mazumdar, 2015). Basicity of some low molecular weight carbonyl compounds towards ICl was studied by Lamsabhi et al. (Lamsabhi, Bouab, Esseffar, Alcami, Yanez & Abboud, 2001) who reported that the carbonyl-ICl interaction in the prepared complexes was essentially electrostatic. Some adducts of N, N’-diacetylpiperazine with I2 and ICl molecules were synthesized and crystal structures investigated by Suponitsky et al. (Suponitsky et al., 2016) who explained the formation of the adducts via C=O---I and C-H---I interactions. However, we found spectral evidences for the formation of an –O—I bond between the carbonyl oxygen of acetyl group and the electrophilic iodine species of ICl reagent. Similar arguments were put forward by Tjarks et. al. (Khalil & Tjarks, 2014) who reacted β-thymidine and 3-substituted β-thymidine analogues with ICl and proposed a reaction mechanism showing an attack by electrophilic iodine to the C-4 carbonyl oxygen of β-thymidine unit or the oxygen atom of 2’-deoxyribose ring forming an oxonium intermediate. Formation of a bond between iodine and polyamides was mentioned by Tonelli et al. (Tonelli, Kotek, Vasanthan & Salem, 2001) who proposed a

. The objective of this study was

reaction scheme showing the formation of

to generate carbonyl groups in the polysaccharide by a route different from the one earlier

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reported by us (Ganie, Ali & Mazumdar, 2015) and attach ICl to the generated –HC=O groups. Another objective was to identify the released iodine species in the medium and understand the chemistry of iodination with ICl and the deiodination in water. We report here the synthesis and characterization of periodate oxidized GA-iodine complexes and the detailed investigation of ICl-aldehyde interaction employing FT-IR, 1H- and

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C- NMR,

DSC, TGA, SEM and UV-Vis spectroscopy. 2. Materials and methods 2.1 Materials Gum arabic (Acacia gum; spray dried LR; molecular weight = 953900 g/mol) was purchased from S.D. Fine Chemicals, Mumbai, India. Sodium meta-periodate, ethylene glycol, ethanol, sodium bicarbonate, potassium iodide, potassium iodate, iodine (resublimed), hydroxylamine hydrochloride, sulphuric acid, hydrochloric acid, sodium thiosulphate and sodium hydroxide were supplied by Merck Specialities Pvt. Ltd, Mumbai, India. Iodine monochloride was prepared in the laboratory following a standard procedure (Roholt & Pressman, 1972). All the chemicals were used as received without any further purification. 2.2 Periodate oxidation of gum arabic Gum Arabic (GA) was oxidized with sodium meta-periodate to different degrees of oxidation by methods reported in the literature (Nishi & Jayakrishnan, 2004; Balakrishnan, Lesieur, Labarre, & Jayakrishnan, 2005) after making necessary changes in the amount of GA, concentration of periodate and the reaction time. The following concentrations of sodium mperiodate (0.117, 0.234, 0.351 and 0.467 mmol/ml in water) were reacted with aqueous solutions of gum arabic (1.0 g was dissolved in 25 ml water). The reactions were performed in dark at 40o C for 24 hours under magnetic stirring and stopped by adding ethylene glycol

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(5.0 ml). The product was precipitated by pouring the reaction mixture in ethanol (150 ml) and the extent of oxidation was estimated by iodometric titration of the residual periodate present in the reaction mixture (Sullivan, Houston, Cervinskas & Gorstein, 1995). In details, 5.0 ml aliquot of the reaction mixture was withdrawn and neutralized with 10 % aqueous sodium bicarbonate solution (10 ml). Iodine was liberated in the mixture by the addition of 20 % aqueous potassium iodide solution (2 ml) and 1M sulphuric acid (5 ml). The solution was kept in dark for 15 min and the amount of excess periodate in the reaction mixture was estimated by titrating the liberated iodine against standard sodium thiosulphate solution (0.1N) using starch as indicator (1 g soluble starch was dissolved in 100 ml water). All the titrations were performed in triplicate. The oxidized derivative was filtered and washed thoroughly with water and ethanol until all the iodine compounds were completely removed (checked with silver nitrate solution). The weights and yields of oxidized gum arabic derivatives obtained using four different concentrations of periodate are: GAL1 (0.837 g, 83.7 %), GAL2 (0.825 g, 82.5 %), GAL3 (0.869 g, 86.9 %) and GAL4 (0.845 g, 84.5 %). 2.3 Determination of aldehyde content The aldehyde content of the oxidized gum arabic samples was determined by converting the aldehyde groups into oximes by Schiff’s base reaction using hydroxylamine hydrochloride (Guo, Ge, Li, Mu & Li, 2014). 0.3 g of the GAL sample was dissolved in 25 ml distilled water and the pH was adjusted to 5.0 by adding 1.0 M aqueous sodium hydroxide solution. Into this was added 20 ml hydroxylamine hydrochloride solution (0.72 mol/l) and the mixture was stirred at 40o C for 4 hours. Titration of the released hydrochloric acid was performed using 1.0 M aqueous sodium hydroxide solution using phenolphthalein indicator and the volume of alkali solution consumed in the titration was recorded as Vo (in litre). The same

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concentration of GA solution (0.3 g dissolved in 25 ml water) maintained at pH 5.0 was used in the blank titration and the volume of 1.0 M sodium hydroxide solution required for the titration was recorded as Vb (in litre). The aldehyde content (% w/w) in the oxidized gum arabic was calculated using the following equation:

Aldehyde Content (%) =

(𝑉𝑜 −𝑉𝑏 )×𝐶𝑁𝑎𝑂𝐻 8×𝑚/𝑀

x 100

where CNaOH = 1.0 M, m is the dry weight of GAL (0.3 g) used in the experiment and M is the approx. molecular weight of the repeating unit, C6O5H10 in the oxidized gum derivative. M has been calculated to be 162 on the basis of a report by Li et al. (Li, Wu, Mu & Lin, 2011). The experiments were performed in triplicate. 2.4 Iodination of oxidized gum arabic with iodine monochloride Iodination of the oxidized gum arabic derivative, GAL4 containing the highest aldehyde content (40.42 %) was carried out using different concentration of ICl. 0.5 g of GAL4 was added to 20 ml ICl solution (0.5 M, 1 M and 2 M) and the mixture was stirred at 50o C for 24 hours. The reaction mixture was next poured into 100 ml ethanol and the solid product was filtered, washed and dried to constant weight. Iodine complexes of oxidized gum arabic prepared with three different ICl concentrations are IGAL1, IGAL2 and IGAL3 and the respective weights and yields are: 0.399 g (79.8 %), 0.421 g (84.3 %) and 0.440 g (88.1 %). A 2.0 M solution of ICl was prepared according to a reported method (Roholt & Pressman, 1972) by adding 22.14 g of potassium iodide and 14.27 g of potassium iodate to 11.6 M hydrochloric acid (50 ml) and water (40 ml) in a 100 ml volumetric flask. The contents were stirred until completely dissolved and the volume was made up to 100 ml. 1.0 M and 0.5 M solutions of ICl were prepared in a similar manner. 2.5 Iodination of oxidized gum arabic with molecular iodine vapour 7

0.3 g of GAL4 was exposed to molecular iodine vapour in a sealed tube that was kept under dark for 24 hours at room temperature. The light brown coloured solid complex, GAL.I2 was characterized to understand the iodine complexation in ICl-oxidied gum arabic adducts. The weight and yield of GAL.I2 are 0.277 g and 92.3 % respectively. 2.6 FT-IR spectroscopy FTIR spectroscopy was performed using TENSOR 37 (Bruker, Billerica, MA, USA) FTIRspectrometer with a horizontal ATR accessory and the samples were scanned in the range of 4000-400 cm-1. Fine powdered form of the samples was used directly for the scan. 2.7 NMR spectroscopy 1

H NMR and 13C NMR spectra were recorded on a Bruker 300 MHz spectrometer (Billerica,

MA, USA) at room temperature using D2O as solvent. 2.8 Determination of iodine content Iodine contents in IGAL1, IGAL2 and IGAL3 were estimated by bromine oxidation method described by Sullivan et al. (Sullivan, Houston, Cervinskas & Gorstein, 1995). In this method iodide, I- is oxidized to iodate, IO3- by bromine water in a slightly acidic solution and then excess bromine is destroyed by the addition of sodium sulphite and phenol. Addition of an excess of iodide liberates elementary iodine, which is then titrated against standardized sodium thiosulphate solution using starch as indicator.

In detail the procedure is as follows: 0.1 g of oxidized gum arabic-iodine complexes, IGALs was dissolved in 10 ml distilled water into which 6 drops of methyl orange indicator was 8

added (the color turned pale orange). The solution was made acidic by adding 2 N sulphuric acid drop wise until the color turned pink. Next 0.5 ml bromine water was added (the solution changed to yellow after a few minutes). 1 % sodium sulphite solution was added drop wise in order to destroy the free bromine liberated in the solution which turned pale yellow in colour. The sides of the flask were washed with water and 2/3 drops of 5 % phenol solution was added to destroy any residual free bromine present in the solution (the solution turned clear). 1 ml of 2 N sulphuric acid and 5 ml of 10% potassium iodide solution were next added to the clear solution which was titrated against 0.005 N sodium thiosulphate solution using freshly prepared 1 % starch solution as the indicator. Available iodine (% w/w) was calculated using the following equation:

Available Iodine (%) =

NST ×VST ×126.9×100 V×ρ

NST is the strength of sodium thiosulphate solution (0.005 N), VST is the volume of sodium thiosulphate solution used (in ml), 126.9 is the equivalent weight of iodine, V is the sample volume (in ml) and ρ is the density of iodine (4.98 g/ml). 2.9 Measurement of viscosity Viscosity measurements of GA, oxidized GA (degree of oxidation 19.68- 50.19%) and the iodine complexes (iodine content 6.11-10.19 %) were conducted at 30o C to know the effects of (i) oxidation and (ii) iodination of oxidized GA on the intrinsic viscosity. 10% stock solutions of GA, GAL derivatives and iodine complexes were prepared in distilled water and the respective stock solutions were diluted to prepare 1%, 2%, 3% and 4% solutions. Flow times for water (t0) and the solutions (t) were determined in an Ostwald viscometer and graphs of reduced and inherent viscosity against concentration were plotted. Double

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extrapolation of the plots to zero concentration was used to determine [η], the intrinsic viscosity from the intercept. 2.10 Scanning electron microscopy with energy dispersive X-ray spectrometry Scanning electron micrographs were recorded with EVO 50 series SEM instrument (Zeiss, Germany). Powdered samples were mounted on aluminium stubs by means of double-sided adhesive carbon tape and coated with gold. The EDS spectrum was obtained with a Si/Li EDS detector at an accelerating voltage of 20 kV. 2.11 Thermal analysis TGA analyses were performed using a Perkin-Elmer thermal analyser (TGA 4000, Massachusetts, USA, Pyris 6 TGA with PyrisTM software V. 11.0.0.0449) at a heating rate of 10o C min-1 from room temperature (38o C) to 700oC under nitrogen atmosphere. DSC scans were obtained using a Perkin-Elmer DSC 6000 thermal analyser (Massachusetts, USA, Pyris 6 TGA with PyrisTM software V. 11.0.0.0449) from 0- 450o C at a heating rate of 10o C min-1 under nitrogen atmosphere. 2.12 X-ray diffraction studies X-ray diffraction pattern of samples was recorded on a Rigaku Ultima IV type X-ray diffractometer (40 kV, X-ray Cu, Tokyo, Japan) with 2ϴ ranging from 10-70 degree. The diffraction angle was scanned at a rate of 8o / min. 2.13 Ultraviolet-visible spectroscopy UV-vis spectral scans of iodinated samples were recorded on a Perkin-Elmer Spectrophotometer (LAMBDA 650, Waltham, MA, USA) to identify the iodine species

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released in water from the samples. The standard plots of various iodine species discussed in our earlier paper (Mazumdar, Chikindas & Urich, 2010) were used as the reference. 3. Results and discussion 3.1 Preparation and characterization of oxidized GA and its iodine complexes Oxidation of adjacent diols with periodic acid or its salt in an aqueous medium is a wellknown reaction named Malaprade oxidation (Wang, 2010). This reaction results into the cleavage of C2-C3 or C3-C4 bond of sugar residues and introduction of a large number of aldehyde groups in the polymer chains. In the present work, gum arabic was oxidized using different concentrations of sodium meta-periodate to obtain 19.68-50.19 % oxidised gum (the degree of oxidation represents the percentage of the total monosaccharide units which reacted with periodate to undergo oxidative degradation) and this corresponded to 5.15-40.42 % aldehyde contents in the dialdehyde-gum samples. The experimental data presented in Table 1 indicate that as the periodate concentration increased, greater extents of oxidation occurred resulting into higher aldehyde contents in the oxidized gum. The sample with the highest number of aldehyde groups (40.42 %) was treated with different concentrations of ICl (0.5 M, 1.0 M and 2.0 M). As the ICl concentration was increased, iodine content of the synthesized complexes was experimentally measured to be increasing from 6.11 to 11.72 % indicating for the first time, in this investigative study, the quantitative aspect of ICl addition to aldehyde groups in oxidized GA. The effect of ICl concentration on iodine content of the produced complexes was investigated by reacting GAL4 with three different concentrations of ICl (0.5-2.0 M). In other words, the extent of interaction between ICl and aldehyde groups increased quantitatively in IGAL2 and IGAL3 as more aldehyde groups were available for reaction with ICl resulting in an increase in the iodine content. The highest degree of oxidation that could be achieved with gum arabic was ~50 %. The ~50 % oxidized gum

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containing 40.42 % aldehyde content reacted with ICl producing a complex with the maximum iodine content (11.72 %). To know the percentage of aldehyde contents that reacted with ICl, we determined the percentage of unreacted aldehyde groups in each sample of the iodinated oxidized gum by the titrimetric method employed earlier. The results indicated that the proportions of aldehyde groups that complexed with ICl in IGAL3, IGAL2 and IGAL1 were 86.64, 73.25 and 56.98 % of the total aldehyde groups present in GAL4. It can thus be concluded that most of the aldehyde groups generated in the ~50 % oxidized gum reacted with ICl (unreacted aldehyde content in IGAL3 = 5.40 %) to have an iodine content of 11.72 %. The iodinated derivatives, IGAL1, IGAL2 and IGAL3 showed much better water solubility as compared to the parent oxidized GA. Interestingly though, the solubility of GAL.I2 in water was not as high as IGAL. This observation suggested that ICl interaction with GAL was different from that between molecular iodine, I2 and GAL. Table 1 Sample

Periodate concentration (mmol)

Yield (%)

Degree of oxidation (%)

Aldehyde content (%w/w)

Iodine content (% w/w)

GAL1

1.17

83.70

19.68

5.15

-

GAL2

2.34

82.55

24.60

18.16

-

GAL3

3.50

86.97

39.30

30.73

-

GAL4

4.67

84.51

50.19

40.42

-

IGAL1

-

79.81

-

17.56

6.11

IGAL2

-

84.33

-

10.81

8.91

IGAL3

-

88.11

-

5.40

10.01

12

GAL.I2

-

92.31

-

-

10.19

Fig 1(a) presents the FTIR spectra of unmodified GA and oxidized GA with different aldehyde contents. GA showed the following characteristic absorption bands: (i) – OH stretching at 3000-3500 cm-1 (ii) – CH2 stretching at 2927 cm-1 and (iii) – CO asymmetric stretching at 1600 cm-1. The peak at 1417 cm-1 was attributed to the skeletal motions of the carbon rings, --CH and –CH2 wagging vibrations (Farooq et al. 2017). The changes in GA structure after oxidation was observed in the form of new absorption bands appearing at 1728 and 881 cm-1 which corresponded to C=O stretching vibrations of dialdehyde and hemiacetal structures respectively. Stefanovic et al. (Stefanovic, Jakovljevic, Gojgic-Cvijovic, Lazic & Vrvic, 2013) reported similar features in the IR spectra of oxidized GA. The intensity of 1728 cm-1 peak that appeared as a shoulder in GAL spectra increased as the degree of oxidation increased from 19.6 to 50.1 %. The oxidized gum derivative with the highest aldehyde content (40.4%) was reacted with ICl to synthesize iodine complexes. The electrophilic iodine in I+Cl- was expected to attach to the carbonyl oxygen of dialdehyde forming -C–O—I structure. Fig 1(b) presents the FTIR spectra of iodine-GAL derivatives containing different iodine contents. In IGAL, the carbonyl absorption peak shifted from 1728 to 1748 cm-1 as a result of an electronic repulsion between non-bonding electrons on oxygen and iodine atoms which clearly showed the involvement of carbonyl group in a reaction with electrophilic iodine species, I+. The intensity of C=O absorption at 1748 cm-1 decreased with increase in ICl concentration confirming iodination of more and more aldehyde groups. This fact was also supported by the iodine content values in the samples, IGAL1, IGAL2 and IGAL3, estimated by iodometric titration (Table 1). New absorption bands appeared at 1716 cm-1, 1530 cm-1, 1052 cm-1 and 786 cm-1 which were attributed to C—O—I and C—Cl vibrations. Typical C—Cl absorption 13

occurs in the broad region between 850 and 550 cm-1 and CHCl wagging is observed near 1083 cm-1 (Silverstein, Webstar & Kiemle, 2005). In order to understand the ICl-aldehyde interaction better, an iodine derivative of GAL was prepared using molecular iodine (I2) vapor and IR spectra of IGAL3 and GAL.I2 were compared [Fig 1(c)]. FTIR spectra of GAL.I2 and IGAL3 clearly bring out the difference in attachment of iodine to the carbonyl groups when I2 and ICl are used respectively. In GAL.I2,

the carbonyl absorption band

appears at a lower frequency (1732 cm-1); but the most important factor was the absence of C—Cl band at 786 cm-1. Figure 1.1

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Figure 1.2

Figure 1.3

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The following conclusions were drawn from the IR studies: (i) both I2 and ICl attacked carbonyl groups in GAL (ii) there was the formation of C—Cl bond when ICl was used (iii) no such bond was formed in the case of I2 addition to GAL (iv) there was no change in the hemiacetal structure of dialdehyde-gum and (v) though the iodine contents in IGAL3 and GAL.I2 were almost the same, their IR absorptions were not similar due to different chemical environment around the carbonyl groups. Figure 2(a)

GAL, 1H-NMR (300 MHz, D2O) δ 9.67(s. 1H), 8.27 (), 7.73 (), 4.70-4.41(m, 8H), 3.22 (s, 1H), 2.44 (s, 2H), 2.29 (d, J = 39.4 Hz, 1H), 2.08-1.96 (3, 2H), 1.51(d, J = 9.0 Hz, 0H) GAL, 13C-NMR (300 MHz, D2O) δ 107.04 (s, 2C), 72.11 (s, 2C), 62.55 (s, 1C), 16.72 (s, 1C) IGAL, 1H-NMR (300 MHz, D2O) δ 4.83- 4.59 (m, 8H), 4.28 (s, 2H), 3.72 (s, 1H), 3.65, 1.50-1.19 (m, 2H) IGAL, 13C-NMR (300 MHz, D2O) δ 175.12 (s, 1C), 170.23 (s, 2C), 107.04 (s, 2C), 103.68 (d, 1C), 92.17 (s, 2C), 77.05-76.83(m, 6C), 72.11 (s, 3C), 62.55 (s, 1C), 16.72 (s, 1C)

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Figure 2(b)

Figure 2(c)

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Figure 2(d)

Chemical shifts (ppm) of GA protons in the 1H-NMR spectrum [presented in fig 2(a)] were compared with that reported by McIntyre and co-workers (McIntyre, Ceri & Vogel, 1996) and a satisfactory matching of the signals was observed. In the proton spectrum of oxidized gum (GAL) shown in fig 2(b), a small but prominent peak at 9.67 ppm appeared that was assigned to the aldehydic protons. Observation of a signal from the aldehydic proton of oxidized GA at 9.30 ppm was reported by Stefanovic et. al (Stefanovic, Jakovljevic, GojgicCvijovic, Lazic & Vrvic, 2013) but they obtained NMR only after a mild acid hydrolysis of GA followed by its oxidation. The complete disappearance of aldehydic proton peak in IGAL [fig 2(c)] and the appearance of a new peak at 5.4 ppm reasonably explained the following facts: (i) aldehyde groups of GAL (present both in open and hemiacetal forms) took part in

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the reaction with ICl (ii) the new peak indicated the presence of a proton attached to carbon bearing chlorine through the formation of –CHCl groups. The

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C-NMR shifts of gum arabic (not shown here) consisted of single peaks at 16.7 ppm

(C-6, Rhap) and 61.4 ppm (C-6, C-5, Galp-Rhaf), a bunch of peaks at 100.8-110 ppm (C-1) and a single peak at 175.1 ppm (C-6, Glap A); the spectrum matched that reported in the literature (Tischer, Gorin & Iacomini, 2002). A clear

13

C-NMR spectrum of GAL could not

be obtained due to poor solubility of the compound in D2O. It showed a poorly resolved carbonyl signal around 175-180 ppm (not shown here). A change of solvent to DMSO-d6 to obtain a satisfactory

13

C-NMR spectrum did not produce a better result. IGAL which was

soluble in D2O showed a new peak at 170.6 ppm [Fig 2(d)] probably due to the attachment of chlorine to the carbonyl carbon (-Cl-C=O). 3.2 Scanning electron microscopy Morphological examination of GA and its oxidized products was carried out with the help of their scanning electron micrographs presented in Figs. 3(a) and (b). Gum arabic powder (a) showed globular particles with dents on the surface, a feature that changed completely after oxidation. Periodate oxidised gum arabic sample (b) had homogenous surface with irregular smaller sized particles appearing topologically. The surface morphology changed drastically after iodination with molecular iodine vapour and iodine monochloride. Iodine vapour treatment resulted into porous surface with small rod like crystals appearing on the cracked structure [Fig. 3(c)]. Iodination with iodine monochloride displayed a unique morphological feature where the surface showed the presence of small regular cubic crystals uniformly scattered over the top [Figs. 3(d)-(f)] and the crystal size was found to enlarge with increasing concentration of iodine monochloride reagent. The particles/crystals size as determined by SEM is as follows:

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IGAL1: 974.3 nm; IGAL2: 1.683 - 2.692 µm; IGAL3: 5.410 – 7.455 µm and GAL.I2: 801.5 nm – 2.323 µm From the EDS spectrum [Fig 3(g)] it was obvious that both iodine and chlorine were present in the iodine complex showing signals around 2.6 and 3.9 KeV for chlorine and iodine respectively. Figure 3

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(g) 3.3 Thermal behavior TG thermograms and DTG curves of the samples are presented in Figs. 4(a) and (b) respectively and it is evident that the thermal degradation of GA, oxidized GA and the iodinated samples occurred in two stages. The first stage weight loss (9.1%) happened in unmodified gum arabic between 56.7-141.6o C due to the release of absorbed and hydrogen bound water (Zohuriaan & Shokrolahi, 2004); the second stage showed a loss of 62.2% between 293.4-343.5oC due to the polysaccharide degradation that finds mention in the literature (Nishi & Jayakrishnan, 2007). Thermal stability of oxidized GA did not change much from the parent gum though the first stage decomposition started at a lower temperature in the former, 50.4o C and continued up to 75.3o C with 6.9% weight loss. Integral procedural decomposition temperature (IPDT) is derived from graphical integration of the thermogravimetric analysis of a polymer and is used here to compare the thermal properties of GA and its derivatives. IPDTs (S1) calculated using Doyle’s method (Doyle, 1968) showed slightly lower value for GAL than GA and this was attributed to the opening of sugar rings during oxidative cleavage reaction as discussed by Nishi et al. (Nishi & Jayakrishnan, 21

2007). IDT1 represents the temperature that corresponds to the beginning of mass loss in the first phase of thermal decomposition of a polymer. GAL.I2 showed IDT1 (initial decomposition temperature) at a further lower temperature of 46.6o C probably due to the volatilization of free iodine molecules entrapped in the polymer matrix (Ahmad, Mazumdar & Kumar, 2013). This is not surprising since iodination was performed using molecular iodine vapour. The iodine complexes synthesized using ICl were found to be thermally more stable than GAL.I2. For IGAL1 (lowest iodine content), first stage decomposition started at 93.2o C and continued till 200.2 o C losing 7.1 % mass while the second and major weight loss (64.9%) occurred between 334.1 and 469.0o C. Within the series of IGAL, thermal stability was observed to be increasing with the iodine content of the samples; both IDT1 and IDT2 increased from 93.2o C to 200.2o C and from 334.1o C to 400.4o C respectively; the calculated IPDT values also supported this trend (S2). This is possibly due to the increase in intermolecular polar interactions present in the samples containing Cl-HC-O-I groups. The overall observations made from the thermal analysis are: (i) oxidized GA has slightly lower thermal stability than pure GA (ii) iodine vapour treated sample has lower thermal stability than ICl treated iodine complexes because of the different nature of iodine attachment and (iii) within IGAL, the thermal stability increased with higher iodine content in the samples.

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Figure 4(a)

Figure 4(b)

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Thermal behaviour of complex polysaccharides has been reported to be irregular and changing with the structural features and functional groups present in the polysaccharide and different moisture contents (Bothara & Singh, 2012). Fig. 5 shows the DSC curves of GA, GAL, GAL.I2 and IGAL samples. GA showed a broad endotherm at 78.29o C and an exothermic peak at 316.40o C. A similar DSC scan for GA was reported by us and the peaks were related to the melting and partial thermal decomposition of the complex polysaccharide consisting of several sugar units present as branches and sub-branches (Ganie, Ali & Mazumdar, 2015). Oxidized GA showed the endotherm at a slightly higher temperature (84.28o C) and the exotherm at 295.58o C probably due to the formation of dialdehyde structures and consequent crosslinking and thus raising the glass transition temperature (Zhang, Wang, Zhang, Yang & Wang, 2010). Iodinated derivatives i.e. GAL.I2 and IGAL showed the endotherm at 72.22 and 52.53o C and the corresponding exotherm at 250.11 and 183.55o C respectively. Lowering of Tg in the iodinated derivatives could be attributed to iodine desorption from the complexes. The much lower Tg observed in IGAL supported the proposed structural differences in the two types of iodinated derivatives.

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Figure 5

3.4 X-ray diffractions XRD measurements were performed to follow the changes taking place in the crystalline region of the natural gum upon oxidation and further modifications. The diffraction pattern of GA, GAL and IGAL are presented in S2. GA is a highly branched polysaccharide which showed a lack of crystallinity as suggested by a single wide peak at 2θ = 19.88o in the spectrum. A similar XRD pattern for natural GA was reported by Almuslet et al. (Nafie, Almuslet Magied & Muhgoub, 2012). Upon oxidation this peak reduced its intensity and became broader due to the opening of the glucopyranose rings and destruction of their packing (Lindh et al., 2014). The iodine complex, IGAL showed a completely different XRD pattern with distinct peaks at 2θ = 28.5, 40.6, 50.2 and 66.5o. This confirms the structural changes occurring in oxidized gum after interaction with ICl.

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3.5 Intrinsic viscosity Intrinsic viscosity of oxidized GA samples ranged between 0.88- 1.66 dl/g in comparison to 0.86 dl/g of pure gum. The results (presented in S3) show that [η] rose with degrees of oxidation suggesting a probable stiffening of chains in the polysaccharide by the opening of sugar units and forming intra-molecular crosslinks between dialdehyde groups; the higher the aldehyde content in GAL the greater would be the extent of crosslinking. Ma and Yu (Ma & Yu, 2012) reported the formation of such crosslinking in dialdehyde-starch. [η] decreased in iodine complexes of the oxidised gum probably due to the breakdown of crosslinks and formation of -C— O—I linkages by the reaction between aldehyde groups and ICl molecules. 3.6 UV-vis spectroscopy Fig.6 shows the UV-Vis spectra of IGAL complexes in water. All the compounds showed a sharp prominent peak at 225 nm indicating the release of iodide ions (I-). The intensity of the peak was found to be increasing with the iodine content in the samples. IGAL3 containing the highest iodine content showed two weak intensity peaks at 285 and 350 nm that corresponded to tri-iodide ions (I3-). The liberation of small amount of I3- ions was probably possible due to the hydrolysis of ICl molecules, electrostatically attached to carbonyl groups (-C=O---ICl) in IGAL, as described by the following reactions (Philbrick, 1947).

The UV-Vis spectrum of GAL.I2 consisted of a small intensity peak at 225 nm for I- along with two broad peaks around 285 and 350 nm for I3-. The observed difference in I- intensity in IGAL and GAL.I2 confirms the suggested attachment of different iodine species in these two complexes (Scheme 1).

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Figure 6

27

Reaction scheme

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4. Conclusions The dialdehyde groups generated in gum arabic via periodate oxidation were used to synthesize iodine complexes of oxidized GA by their interaction with different iodine forms. The results suggested that electrophilic addition of an ICl molecule to the –CHO group took place in the case of iodination with ICl, whereas in the other reaction, molecular iodine, I2 was electrostatically attached to the carbonyl groups in the gum derivative. We differ with the existing literature reports (Lamsabhi, Bouab, Esseffar, Alcami, Yanez & Abboud, 2001; Firdoussi, Esseffer, Bouab, Lamsabhi, Abboud, Mo & Yanez, 2003; Firdoussi et al., 2004) that ICl forms CT complexes with carbonyl compounds. Since the iodination mechanism and the nature of bound iodine species to the polysaccharide complexes were different, the released form of iodine in water was also observed to be different as detected by UV-Vis spectroscopy. The ICl modified complex predominantly released I- (iodide) ions whereas the iodine vapour treated complex mostly released I3- ions. The mechanism of reactions during synthesis of iodine complexes and release are based on the experimental and spectral data. Iodide is the reduced form of iodine that is preferentially taken up by the thyroid gland for producing thyroid hormones. The iodine supplements available on the market to treat iodine deficiency are potassium iodide, Lugol’s solution of iodine (10 % potassium iodide, 5 % elemental iodine and 85 % water) and nascent iodine. Hence an iodide releasing edible gum would have better prospects in iodine nutrition. Acknowledgements Two authors (A. A. and S. A. G.) would like to thank University Grants Commission, New Delhi, India for Research Fellowships.

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Figure captions, tables and schemes Table 1. Yield, extent of oxidation, aldehyde and iodine contents Fig 1.1 FTIR spectra of (a) GA, (b) GAL1 (c) GAL2 (d) GAL3 and (e) GAL4 Fig 1.2 FTIR spectra of (a) GAL4 (b) IGAL1 (c) IGAL2 and (d) IGAL3 Fig 1.3FTIR spectra of (a) GAL4 (b) GAL.I2 and (c) IGAL3 Fig 2(a) 1H NMR spectrum of GA (D2O) (b) 1H NMR spectrum of GAL (D2O) (c) 1H NMR spectrum of IGAL (D2O) (d) 13C NMR of IGAL (D2O) Fig 3. SEM images of (a) GA (b) GAL4 (c) GAL.I2 (d) IGAL1 (e) IGAL2 (f) IGAL3 and (g) EDS spectrum of IGAL Fig 4 (a) TGA and (b) DTG curves of GA, GAL, GAL.I2, IGAL1, IGAL2 and IGAL3 Fig 5. DSC curves of GA, GAL4, GAL.I2 and IGAL1 Fig 6. UV-Vis spectra of IGAL1, IGAL2, IGAL3 and GAL.I2 Reaction scheme: Schematic representation of (a) periodate oxidation of GA and iodination of GAL with ICl (b) release of iodide ( I-) ions (c) iodination of GAL with molecular I2 vapor

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