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Alternative approach to synthesize methylated chitosan using deep eutectic solvents, biocatalyst and ‘green’ methylating agents Prachi S Bangde, Ratnesh D Jain, and Prajakta Dandekar ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00653 • Publication Date (Web): 09 May 2016 Downloaded from http://pubs.acs.org on May 9, 2016

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ACS Sustainable Chemistry & Engineering

Alternative approach to synthesize methylated chitosan using deep eutectic solvents, biocatalyst and ‘green’ methylating agents Prachi S. Bangdea, Ratnesh D. Jainb* and Prajakta Dandekara* a

Department of Pharmaceutical Sciences & Technology, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai−400 019, India. b Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai−400 019, India

Corresponding Authors E-mail address: [email protected]. Phone: +91-22-3361-2221 Fax: +91-22-3361-1020 (Dr. Prajakta Dandekar) E-mail address: [email protected]. Phone: +91-22-3361-2029 Fax: +91-22-3361-1020 (Dr. Ratnesh Jain)

ABSTRACT: Conventional synthesis of N-methylated chitosan involves use of organic solvents in alkaline conditions, using methyl iodide as the methylating agent. However, the method does not result in selective N-methylation and is known to cause heavy polymer scission. In this investigation we have reported alternative ‘green’ approaches for methylated chitosan synthesis. Two types of Deep Eutectic Solvents (DESs) viz. DES(Urea) and DES(Gly), either alone or in combination with other solvents, were screened as media for facilitating methylation of chitosan. Our results indicated that DESs mediated selective N-methylation in absence of NaOH, with no polymer scission, when compared with the reported methods. 1H NMR spectra confirmed selective N-methylation in case of products obtained using DES(Urea), while that obtained using DES(Gly) demonstrated some O-methylation. Another green method investigated unexplored property of biocatalyst lipase for methylating chitosan in presence of ‘green’ methylating agents in DESs systems. Furthermore, lipase from Burkhorlderia species exhibited ability to methylate chitosan polymer while the enzyme from Candida Antartica failed to methylate the polymer. Our investigation also confirmed the possibility of using dimethyl carbonate as a benign methylating agent.

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KEYWORDS: Trimethyl Chitosan (TMC), Deep Eutectic Solvents (DESs), N-methylation, biocatalyst lipase, green methylating agents. INTRODUCTION The demand for biopolymers as drug delivery vehicles is continuously escalating due to their biocompatibility, biodegradability and ability to target or control the release of encapsulated therapeutic agents. Chitosan, an abundant biopolymer has proved its utility in biomedical, pharmaceutical, food and textile industries.1 Although of immense potential, the material possesses drawback of selective solubility at acidic pH, which has stimulated scientists to investigate variants of this biopolymer. One widely studied derivative is N,N,N-trimethyl chitosan (TMC), which possess a permanent positive charge on its amine group. The polycationic nature of this polymer imparts it higher solubility at neutral and alkaline pH, while enhancing its interactions with biological membranes by opening the tight intracellular junctions and increasing the paracellular uptake of therapeutic molecules.2,3 Methylated chitosan is traditionally synthesized using organic solvents like acetic acid, N-methyl-2pyrrolidone (NMP), dimethyl sulfate (DMS), dimethyl formamide (DMF) using methyl iodide as the methylating agent. Most of these methods suffer from shortcomings including lack of selective Nmethylation, environmental hazards of reagents, involvement of tedious procedures and possibility of scission of the parent polymer or poor yield of the final product.

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This has demanded alternative,

environmentally-friendly strategies to synthesize TMC. Very recently green chemistry has garnered much attention due to its ability to provide newer processes that minimize the use and generation of hazardous substances. The focus of these methods is on utilizing cheap, renewable and environmentally safe starting-materials. In this regards, deep eutectic solvents (DESs) are seen as favorable alternatives to the conventional organic solvents.9,10 Apart from renewability, DESs exhibit other advantages including biodegradability, non-toxicity, large-scale


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availability, having very low vapour pressure and high thermal stability, low flammability and easy reusability, as compared to the conventional organic solvents.11 These benefits prompted the use of DESs for our investigation. Further, we have demonstrated the role of enzymes in methylation. We utilized lipase enzyme, which is a promiscuous enzyme, known for its catalytic activity at a broad range of pH, at varying temperatures and for diverse substrate types (without cofactors).12 Although lipases have been primarily reported for use in trans-esterification, esterification, hydrolysis, aminolysis, etc. reactions

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, it has been also

reported to catalyze alkylation reactions by a few researchers owing to its non- specific activity.16 We further attempted to reduce the toxicity of traditional methylating agents like methyl iodide by using dimethyl carbonate as a ‘green’ methylating agent. Many researchers have used this chemical as methylating agent for diverse reactions like O-methylation, N-methylation C-methylation and Smethylation. 17-19 Such combination of reaction media, biocatalyst and methylating agent to conduct methylation of chitosan has been utilised for the first time in this investigation, to the best of our knowledge. Our process has the potential to overcome the drawbacks of traditional TMC synthesis and can provide an alternate, safe approach for scalable synthesis of TMC. RESULTS AND DISCUSSION In this current study, methyl groups were introduced on the amine groups of chitosan to yield trimethylated, di-methylated and mono-methylated polymer by different ‘green’ approaches. One of the approaches focused on employing DESs as the reaction media, alone and in combination with other solvents (Table 1) with methyl iodide as methylating agent. Further, another method was focused on employing biocatalyst lipase with green methylating agents like dimethyl carbonate (DMC) and methanol. The parent polymer, chitosan, was found to be readily soluble in water, DESs, and their combinations with water and DMF. The general reaction scheme is depicted in Scheme 1. As a control, ACS Paragon Plus Environment


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TMC was synthesized by a reported chemical method, which involved reaction of chitosan in DMF/water mixture with methyl iodide as methyl donor group in alkaline conditions, at room temperature (30˚C) for 48h.8 The reaction was conducted to mediate methylation of the amino groups of chitosan at the C-2 position, to form quaternary, amino groups, along with some degree of di- and mono-methylated polymer. The final product (yield ~ 50%) was found to be soluble upto pH 9, unlike the parent polymer which is insoluble at alkaline pH. Also the methylated chitosan powder was observed to be hygroscopic in nature. We followed a similar approach using DESs as reaction medium. Higher methylation efficiency and reduced polymer scission was observed in reaction conducted using DESs, in comparison to reported method. Also, it was found that lipase reaction in DESs in water, with DMC as methylating agent, resulted into N-methylation and O-methylation of chitosan polymer with increase in molecular weight (The preparation of DESs, synthesis and characterization of methylated chitosan are given in the Supporting Information). Table 1: List of DESs solvent system investigated in this work DES(Urea)a solvent systems

DES(Gly)b solvent systems

Ratio (v/v)

DES(Urea)

DES(Gly)

0:0

DES(Urea)+H2O

DES(Gly) +H2O

1:1

DES(Urea)+DMF

DES(Gly) +DMF

1:1

DES(Urea)+H2O+DMF

DES(Gly) +H2O+DMF

1:2:2

a

represents solvent system using DES(Urea) which is synthesized using choline chloride and urea in 1:2 molar ratio. b represents solvent system using DES(Gly) which is synthesized using choline chloride and glycerol in 1:2 molar ratio


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Scheme 1: Schematic representation of methylation reaction of chitosan Characterization of methylated chitosan The presence of various functional groups of the parent polymer and the methyl groups in methylated product was ascertained by FTIR. The FTIR spectrum for polymer showed prominent peaks at wave numbers 1200-1100 cm-1 due to the C-O-C bond on glycosidic linkage, presence of asymmetric angular deformation of C–H bonds of methyl groups at 1475 cm-1, peaks at 1655 and 1320 cm-1 characteristic of N- acetylated chitin, peak at 1500–1630 cm-1 resulting from the angular deformation of N–H bond of amino groups. The last peak disappeared/became weaker in intensity upon methylation due to the occurrence of N-methylation. Further, the methylated chitosan products showed presence of additional peaks at 1630–1660 cm-1 due to quaternary ammonium group and peak at 1415–1430 cm-1 indicating characteristic absorption of N–CH3 bond. The spectra have been included in Figure S1 of the supporting information. Further, 1H NMR was performed to confirm synthesis of TMC from chitosan. The 1H NMR spectrum of chitosan depicted peaks at 3.4–4.5 ppm representing proton of carbon 3, 4, 5, 6, 6′, at 1.9-2.1 ppm due to proton of acetamide group and a peak at 3.0-3.1 ppm due to proton attached to carbon-2. Structural modifications introduced by methylation on chitosan were denoted by formation of new peaks at 2.7-2.8 ppm, indicating mono-methylation; those at 2.9-3.0 ppm, indicating di-methylation and peaks at 3.1-3.2 ppm, confirming tri-methylation. In our investigations, the peaks were up-field with respect to bands ACS Paragon Plus Environment


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reported in literature (mono-methylation - 3.0 ppm, di-methylation- 3.2 ppm and trimethylation - 3.3 ppm).8 The possible reason for this may be that all 1H NMR samples reported in literature were analyzed at higher temperature, which increased vibrational frequency of polymers in solution (by decreasing the solution viscosity and making the polymers more available for magnetic field of 1H NMR). In our analysis, samples were assessed at room temperature and hence the characteristic bands were observed up-field. As per the recorded 1H NMR spectra (Figure S2 of Supporting information), the DD% of chitosan polymer was observed to be 94.53%. Effect of NaOH on molecular weight of methylated chitosan Various researchers have suggested that NaOH is required during methylation of chitosan to maintain its deprotonated state and thus increase probability of attack of methyl groups on amino groups of the polymer. Furthermore, studies suggest that an inorganic base, such as an aqueous NaOH solution, is better than organic bases, since NaOH has a larger pKa than chitosan and hence neutralizes the hydroiodic acid (HI) generated during reaction. Also, various studies state that high concentrations of NaOH are able to yield higher amounts of substituted polymer, but with simultaneous O-alkylation, which decreases the solubility of the reaction product. Moreover, the use of NaOH has been known to enhance polymer degradation.6, 20 Thus in our investigation, we studied the influence of NaOH on the molecular weight of the resulting polymers to assess any chain degradation. Various concentrations of NaOH viz 0.5, 5, 10, 15, 20% (w/v) were evaluated. The aim was to identify that concentration of NaOH, which would result in minimum degradation of the parent polymer. Molecular weight of chitosan and its products were determined by Gel Permeation Chromatography (GPC). Molecular weight of the native chitosan polymer was evaluated as 16kDa. Upon treatment with NaOH, it was observed that the concentration of NaOH was directly related to polymer scission of chitosan polymer (Figure 1). Minimum polymer scission was observed when concentration of NaOH was 0.5% (w/v), while a concentration of 20% (w/v) resulted in maximum scission. Hence the former


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concentration of NaOH was employed for further reactions. Similar effect of NaOH on chitosan is well reported in literature.21

Figure 1: Effect of NaOH concentration on molecular weight of chitosan

Synthesis of methylated chitosan in DESs: effect on DQ and molecular weight The average degree of substitution (DS) is the most important characteristic of TMC for its applications in pharmaceutical field as it governs its physicochemical properties.22 1H NMR spectroscopy is the most commonly reported technique to determine DS of a polymer. Table 2 represents the DS values obtained for methylated-chitosan synthesized using DESs and its different combinations. Reported chemical method to synthesize methylated chitosan (method described in the supporting information) was performed in order to investigate the influence of repeated methylation on the degree of quaternization (DQ) and molecular weight of methylated chitosan as shown in Figure 2. It was seen that the DQ increased with increase in number of methylation steps, but resulted in O-methylation. From Table 2 it is clear that when a single methylation reaction was conducted, O-methylation was not observed. While, repeated methylation of the methylated product yielded 17% (second methylation) and 86.27% (third methylation) of the O- methylated chitosan, respectively. Influence of DESs or their combinations during the reaction was thoroughly evaluated. It was observed that the product synthesized using DES(Urea) and DES(Gly), in combination with DMF, possessed a



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good DQ (12.05% and 18.68% respectively; Table 2), which may be due to interaction of DMF with hydroxyl groups of chitosan, making amine groups more available for reaction with methyl iodide.8 Methyl iodide and sodium hydroxide have been reported to be the two most efficient chemicals for methylation of chitosan. Therefore, effect of DESs on DQ was evaluated after eliminating NaOH from the reaction mixtures. Effects of reaction media, with and without NaOH on degree of methylation and molecular weight of the final product have been shown in Table 2 and Figure 3, respectively. From Table 2, it is evident that combinations of DESs with 0.5% NaOH (Samples 4-11) resulted in enhanced N-methylation, as compared to reported chemical methylation reaction (Sample 1). These results indicate selective N-methylation in presence of DESs, as against reported method (Table 2, Samples 2 and 3).

Figure 2: Representation of molecular weight and DQ with single, double and triple methylation reactions (where single methylation –sample 1, double methylation – sample 2, triple methylation – sample 3)

Figure 3: Molecular weight of methylated chitosan using various solvent systems (numbers on x-axis represents solvent systems as indicated in Table 2 and 3)


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Table 2: Reaction conditions for synthesis of methylated chitosan in different solvent systems in presence of NaOH with number of reactions carried out and degree of substitution determined by 1 H NMR % DQd % DDie

% DMf % DOg

6

5.50

38.52

26.23

0

DMeb

6

11.06

74.81

22.53

17.00

DMF:H2O

TMc

6

38.18

73.97

1.84

86.27

4

DES(U)

SM

4

1.33

23.84

28.98

0

5

DES(U) + H2O

SM

9

8.67

39.94

21.28

0

6

DES(U) + DMF

SM

12

12.05

55.94

16.61

0

7

DES(U) +H2O +DMF

SM

12

5.58

37.94

19.53

0

8

DES(Gly)

SM

12

12.26

66.31

16.82

0

9

DES(Gly) + H2O

SM

9

7.11

43.09

42.15

0

10

DES(Gly) + DMF

SM

6

18.68

62.99

23.76

0

Samples number*

Polymer from Solvent Reaction 0.5% NaOH system steps (mL)

1

DMF:H2O

SMa

2

DMF:H2O

3

DES(Gly) +H2O +DMF SM 6 6.84 43.50 23.16 0 11 b c d Single methylation (SM) step. Double methyl (DM) step. Triple methylation (TM) step. Degree of quaternization (DQ). e Degree of Di-methylation. f Degree of mono-methylation. g Degree of Omethylation (DO) determined by 1H NMR. * Sample number represents methylated chitosan derived from respective solvent systems. a

Table 3: Reaction conditions for synthesis of methylated chitosan in different solvent systems without NaOH with number of reactions carried out and degree of substitution determined by 1H NMR 0.5% NaOH (mL)

% DQa

% DDib

% DMc

% DOd

SMe

-

0.61

39.44

2.75

0

DES(U) + H2O

SM

-

2.66

67.01

12.08

0

14

DES(U) + DMF

SM

-

2.66

74.09

12.0

0

15

DES(U) +H2O +DMF

SM

-

4.65

13.96

66.83

0

16

DES(Gly)

SM

-

0.21

36.69

0

38.18

Samples number *

Polymer from solvent React ion system steps

12

DES(U)

13



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17

DES(Gly) + H2O

SM

-

0.66

26.12

0.66

24.50

18

DES(Gly) + DMF

SM

-

0.35

34.35

8.99

34.78

19

DES(Gly) +H2O +DMF

SM

-

0

36.20

4.0

44.01

a

Degree of quaternization(DQ). b Degree of Di-methylation. c Degree of mono-methylation. d Degree of O-methylation (DO) determined by 1H NMR. e Single methylation (SM) step. * Sample number represents methylated chitosan derived from respective solvent systems.

Also it was observed the synthesis conducted in DESs with DMF and water, in absence of NaOH, resulted in methylated polymers having lower DQ (samples 12-19) as compared to DQ obtained in presence of 0.5% NaOH (samples 4-11). Moreover, in absence of NaOH, DES(Urea) and its combinations (samples 12-15) resulted in a higher DQ as compared to DES(Gly) and its combinations (sample 16-19). This may be due to reduction in the protonation and concomitant increase in reactivity of the amine group of chitosan towards methyl group owing to higher proton affinity of DES(Urea) and its combination. Thus in absence of NaOH, DES(Urea) and its combinations can be considered as better reaction media over DES(Gly) due to its mild alkaline nature. The investigation further indicated that methylated chitosan synthesized using DESs, in presence of NaOH (Figure 3, samples 4-11), possessed a lower molecular weight than those synthesized in absence of NaOH (Figure 3, samples 12-19). Also, molecular weight of methylated chitosan obtained by reported method (Figure 3, sample 1) was found to be less than molecular weight of methylated obtained using DESs without NaOH (Figure 3, samples 1219). As evident from Table 3, however, the use of DES(Gly) and its combinations (samples 16-19) resulted in O-methylation in absence of NaOH, which was not observed for methylated chitosan obtained using DES(Urea) and its combinations (samples 12-15). Overall, the observations implied that both O-methylation and polymer scission (evident from reduction in molecular weight of the parent polymer), which were observed with reported chemical synthesis were obviated by using DES(Urea) and its combinations. Moreover, DES(Urea) provided mild alkaline conditions, thus eliminating the need of NaOH during reactions (contrary to reported method), which again yielded methylated product without degrading the parent polymer. ACS Paragon Plus Environment

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Methylation of chitosan using enzymes We employed two different types of lipases viz lipase from Burkholderia species (bacteria) in free form and that from Candida Antartica species (fungus) in immobilized form. These enzymes are known to exhibit non-specific reactions. Activity of these enzymes was assessed in various solvent systems to identify the best reaction medium for methylation. This assay was based on ability of the enzymes to hydrolyse ester bond of substrate p-Nitrophenyl acetate (pNPA) into P-Nitrophenol (pNP) and acetic acid, which was studied by spectroscopy. As seen from Figure 5, that the enzyme Amano Lipase PS from Burkholderia cepacia retained its activity in DES(Urea) and methanol as compared to blank (buffer, 7.2) in absence of DESs and organic solvents as control. Although the activity of enzyme in DES(Gly) was less compared to blank (buffer, 7.2), but it was observed to be higher than in solvents like DMF-water (1:1, v/v) and DMC. Moreover, reduction in the activity of the enzyme indicated its probable deactivation. DESs and methanol were thus found to retain the enzymatic activity as compared to DMF-water and DMC. Methanol did not affect the enzyme activity and hence was chosen as the methyl donor for enzymatic reaction. However it was observed that DMC drastically reduced activity of enzyme clearly suggesting enzyme deactivation. On the other hand, the enzyme activity of CAL B (Candida Antarctica Lipase B) was observed to decrease in all the solvents/solvent combinations as compared to the blank, which was attributed to its destabilization.

Figure 5: Enzyme activities for enzyme activity for (A) Amano lipase PS, from Burkholderia cepacia (B) Immobilized CAL B (Candida Antarctica Lipase B)


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Table 4: Enzymatic reaction conditions for synthesis of methylated chitosan using Amano lipase (Burkholderia cepacia) in DES(Urea) solvent system along with degree of substitution determined by 1H NMR and molecular weight by GPC Methylating agent

Mw (kDa) % DQa

% DDib

% DMc %DOd

DMC

198

0

0

0

0

Methanol

62

0

35.86

0

10.11

a

Degree of quaternization(DQ). b Degree of Di-methylation. c Degree of mono-methylation. d Degree of O-methylation (DO) determined by 1H NMR

Scheme 2: Scheme for enzymatic reaction Thus the immobilized enzyme was found to be unsuitable to catalyze the methylation reaction in green solvents and was not used for further reactions. This may be attributed to the fact that the activity of immobilized lipase is hindered due to solvent mediated destabilization. The other possible reason for low activity of immobilized CAL B may result due to poor mass transfer in viscous DESs solvent, thus greatly reducing the activity of the enzyme (The characterization of Lipase enzyme are given in the Supporting Information). This was also experimentally observed when we studied the activity of these enzymes in various solvent systems, based on their ability to convert p-Nitrophenyl acetate (pNPA) to P-Nitrophenol (pNP). As seen from the Figure 5, the free enzyme - Amano lipase PS, from Burkholderia cepacia, was more active in converting pNPA to pNP in DES(Urea) and DES(Gly) than the immobilized CAL B. However, both the enzymes displayed similar activities buffer pH 7.2, which ACS Paragon Plus Environment

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has lower viscosity than DESs. This once again suggested that the low activity of immobilized enzyme in DESs may be contributed by mass transfer limitations in viscous media. Enzymatic methylation using free form of lipase from Burkholderia cepacia in DES(Urea), using DMC as the methylating agent, resulted in di-methylated chitosan as the major product, with simultaneous occurrence of O-methylation. The structural modifications introduced were confirmed by 1H NMR, which clearly indicated the presence of a high intensity peak at 3.0 ppm, which is due to di methylation on amine group of chitosan. The degree of di-methylation was found to be 35.86%. Also O-methylation was observed as indicated by the peak at 3.35 ppm. Further, no methylation was observed in case of product obtained using methanol as methylating agent (as reflected from 1H NMR).

Though a

considerable increase in molecular weight of the product was observed, it was thought to be due to possible cross-polymerization of parent chitosan. Thus, DES(Urea) with Burkholderia cepacia lipase catalyst and DMC as the methylating agent yielded a desirable product. The enzymatic reaction suggested that efficient di-methylation can be conducted at a temperature of 50˚C, adding DMC as the methylating agent in small portions at frequent intervals obviates the need of NaOH that causes polymer scission and methyl iodide that is highly toxic. Without the use of enzyme, the methylating ability of DMC has been explored at a temperature greater than 160 ˚C, much higher that the boiling point of DMC at 90 ˚C.18 Thus our investigation suggested that enzymatic methylation can be conducted at 50˚C, which is a much lower temperature than the boiling point of DMC. FTIR and 1H NMR studies confirmed formation of di-methylated (IR: 1658 cm-1 :N-CH3, 1475 cm-1 : C-H and 1H NMR: 3.0ppm;N-(CH3)2) amino groups on chitosan. This lipase-mediated alkylation by enzyme was thought to occur through generation of methyl groups from DMC, which react with amine and hydroxyl groups of chitosan, with possible release of carbon dioxide and methanol as side-products (Scheme 2).


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CONCLUSIONS Our investigation suggested the potential of bio-based DESs as suitable reaction media for conducting methylation of chitosan. Reaction in DESs with DMF solvent system resulted in maximum degree of substitution. Maximum tri-methylation (12.05%) was achieved using a combination of DES(Urea) and DMF as the reaction medium, while 18.68% using a combination of DES(Gly) and DMF as reaction medium, in a single methylation step. The single methylation reaction with the ‘green solvents’ yielded products comparable to those synthesized chemically after two subsequent methylations. This was noteworthy in terms of reduced organic reactant amounts and reaction time by our synthesis method. Also DES(Urea) without NaOH resulted in N-methylation without affecting molecular weight of the final product, thus obviating need of corrosive chemicals in our reaction. Further, DES(Urea) and DES(Gly) with DMF can be regarded as more efficient than traditional organic solvents in terms of their performance and safety. Another green approach by using biocatalyst was explored; methylated chitosan was obtained using DMC as methyl donor in DESs, in presence of lipase from Burkholderia species. We observed that the source of enzyme and its form also contributed significantly during methylation of chitosan. In our case, immobilized CAL B lipase did not catalyze methylation reaction, which may be due to its destabilization in the chosen reaction media. However, free form of lipase derived from bacterial sources effectively methylated chitosan polymer. Thus the study proved previously unexplored ability of lipase to methylate molecules, in addition to its conventional reactions. ASSOCIATED CONTENT Supporting Information Experimental procedures for preparation of DESs, synthesis of methylated chitosan using DESs and by enzymatic synthesis, compound characterization FTIR, GPC, 1H NMR with FTIR and NMR spectrum


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are available in supporting information. “This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Authors E-mail address: [email protected]. Phone: +91-22-3361-2221 Fax: +91-22-3361-1020 (Dr. Prajakta Dandekar) E-mail address: [email protected]. Phone: +91-22-3361-2029 Fax: +91-22-3361-1020 (Dr. Ratnesh Jain)

Conflict of Interest The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors are thankful to Ramanujan fellowship research grant (SR/S2/RJN-139/2011) and Ramalingaswami fellowship research grant (BT/RLF/Re-entry/51/2011) for financial support. The authors acknowledge Pulorite Private Limited, Pune, India for the generous gift of CAL B lipase. The authors thank Vijay D. Yadav for his technical advice and help during the project. ABBREVIATIONS DDi, Degree of Di-methylation; DESs, Deep eutectic solvents; DES(Gly), Deep eutectic solvent made up of choline chloride and glycerol; DES(Urea), Deep eutectic solvent made up of choline chloride and urea; DM, Degree of mono-methylation; DMe, Double methyl step; DO, Degree of O-methylation; DQ, Degree of quaternization; SM, Single methylation step; TM, Triple methylation step; TMC, N,N,Ntrimethyl chitosan.


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For Table of Contents Use Only Alternative approach to synthesize methylated chitosan using deep eutectic solvents, biocatalyst and ‘green’ methylating agents Prachi S. Bangde, Ratnesh D. Jain and Prajakta Dandekar

SYNOPSIS: A novel green approach has been explored to methylate chitosan polymer in deep eutectic solvents (DESs) replacing organic solvents as reaction medium. Also successfully investigating biocatalyst Lipase and begnin methylating agents as novel green approach to methylate chitosan.


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Alternative Approach to Synthesize Methylated ... - ACS Publications

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