Synthesis and characterization of branched polysaccharides by reaction of cellulose with 2,3,4,6-tetraacetyl-1-bromo- - D - glucopyranoside

Non-naturally branched polysaccharides were prepared homogeneously by reaction of cellulose in N,N -dimethyl acetamide/LiCl and the ionic liquid 1-N -butyl-3-methylimidazolium chloride with 2,3,4,6-tetraacetyl-1-bromo- - D -glucopyranoside in the presence of triethylamine as base. Degrees of substitution up to 0.58 were realized. The samples were soluble in dimethyl sulphoxide and water. NMR spectroscopy and methylation analysis revealed the formation of 1,2-orthoesters. The purity of the products regarding non-bonded sugar molecules was evidenced by advanced NMR techniques (DOSY-and T 2 measurements)


Introduction
Cellulose, one of the most abundant polysaccharides, is the feedstock for many biopolymerbased products.Due to the unique structure, cellulose is insoluble in water and common organic solvents.Soluble materials are obtained by appropriate derivatization reactions that are of interest in many application fields.The pendant groups render the polymer soluble in different types of solvents -of course-depending on the type of substituent and the degree of substitution (DS).Esters (e.g., cellulose acetates, propionates, and mixed derivatives) are soluble in organic solvents, while ethers (e.g., methyl, hydroxyalkyl, and carboxymethyl cellulose) dissolve in water at certain DS.The latter ones are often applied as thickening agents in industrial and household applications and as additive in building materials, paints, drilling fluids, food and personal care products. 1,2Naturally occurring branched polysaccharides like amylopectin, glucoand galactomannans exhibit water solubility even without derivatization. 3Several attempts have been made in order to produce artificially branched polysaccharides.Thus, enzymes have been applied for grafting of glucose onto cellulose yielding materials with improved properties. 4 Grafting of glucose onto powdered cellulose yields colloidal aqueous dispersions useful as sizing or adhesive agent.Glasser et al. have reacted cellulose in N,N-dimethyl acetamide (DMA)/LiCl solution with diethylaminosulfur trifluoride or hydrogen fluoride to introduce long-chain branches by transglycosylation. 5Furthermore, the introduction of monosaccharide moieties into the polysaccharides by using reactive intermediates has been studied since a long time.Usually, 2,3-di-O-protected cellulose derivatives (2,3-O-esters or 2,3-O-phenylcarbamates) are used for the regioselective glycosylation at position 6 by reacting the polymer with acetobromosugars in the presence of both mercuric compounds 6 and strong bases. 7Moreover, sugar orthoesters were reacted with 2,3-di-O-phenylcarbamoyl cellulose and starch. 8,91][12][13] Branched polysaccharides exhibit interesting properties, in particular bioactivity.Thus, the influence of sugar side chains on the biological activity of clinically used polysaccharides was investigated. 14,15Kurita et al. have found that some properties of chitin and chitosan (antimicrobial activity, aggregation ability for bovine serum albumin) are improved after glycosylation reaction with a glucosamino derivative. 16n the course of our ongoing work on water-soluble cellulose derivatives, we have reported results on oxidized cellulose derivatives 17 , cellulosics bearing cationically charged moieties, 18 cellulosics bearing oxocarbonic acid esters of high DS, 19 carboxymethyl cellulose 20 , and cellulose polyelectrolytes prepared via click-reaction. 21or homogeneous chemical modification of cellulose, DMA/LiCl developed by McCormick, is a very versatile reaction medium. 22Moreover, ionic liquids (IL) gained increasing interest in carbohydrate chemistry, 23 in particular for esterification [24][25][26] and etherification. 27However, it was realized that some IL (e.g.1-N-ethyl-3-methylimidazolium acetate, EMIMAc) may lead to side reactions during esterification with carboxylic acids and the anion is included in the chemical modification of the biopolymer.As a consequence, pure cellulose acetate is formed instead of the desired ester. 28n this paper we report the reaction of cellulose with 2,3,4,6-tetraacetyl-1-bromo--Dglucopyranoside under homogeneous conditions in DMA/LiCl and IL without the use of silverand mercury salts to yield soluble cellulose derivatives.The structure of the resulting products was investigated by FTIR-and NMR spectroscopy.

Results and Discussion
The reaction of cellulose was studied under homogeneous conditions using DMA/LiCl as classical solvent and the IL 1-N-butyl-3-methylimidazolium chloride (BMIMCl) and EMIMAc.The dissolved polymer was allowed to react with 2,3,4,6-tetraacetyl-1-bromo--Dglucopyranoside in the presence of triethylamine as acid scavenger (Scheme 1).Precipitation, washing with ethanol, and drying afforded the products as coffee-brown powders.The acetyl group content of selected samples was determined by saponification and titration and used for the calculation of the degree of substitution (DS).
The FTIR spectrum of sample 4 shows all typical structural features of a cellulose derivative (spectrum not shown).Thus, a broad absorption band around 3479 cm -1 indicated the presence of hydroxyl groups as expected with respect to the low DS of 0.58.The band at 2957 cm -1 is attributed to the CH moieties in the molecule.The intensive carbonyl band at 1747 cm -1 evidenced the presence of the acetylated glucose moieties attached to the cellulose backbone.Moreover, the C-O-C valence vibrations could be found at 1245 cm -1 (ester) and 1042 cm -1 (AGU).13   C NMR spectroscopy revealed the presence of glucose moieties attached to the cellulose backbone (sample 2, Figure 1).The signal at 20.8 ppm was attributed to the methyl group of the acetyl moiety at the attached glucose moiety.Various peaks appeared in the range between 60 and 103 ppm that are caused by both the cellulose backbone and the attached acetyl glucose moiety.Unfortunately, it was not possible to distinguish between signals from backbone and the sugar moieties attached.However, a signal at 97.0 ppm is characteristic for the C-1 of a single sugar molecule, while the peak at 103.2 ppm belongs to the carbon atom of position 1.A set of signals at 169.1-170.4ppm was assigned as carbonyl carbon atoms of the acetyl glucose moiety.According to Tsui and Gorin, 29 orthoesters can be formed from 2,3,4,6-tetraacetyl-1-bromo--Dglucopyranoside and hydroxyl compounds.The carbon atom of the orthoester (C-8) could be detected at a chemical shift of 121.4 and 121.7 ppm.Moreover, the peak at 23.4 ppm was accordingly assigned as the corresponding methyl group (C-7).This finding was supported by the 1 H-NMR spectrum of sample 2 (Figure 1) showing signals at 5.81 and 5.75 ppm (H-1'') as well as at 1.75 and 1.65 ppm (H-7, orthoester).Although the peaks of H-1'' and H-7 are not baseline separated, the integral ratio is almost equal to 1:3, and corresponds with the expected value for the orthoester.
The chemical shifts of both the polymer backbone and the acetylated glucose moieties are very similar, and, hence, it was impossible to distinguish between them.Therefore, the 13 C-NMR spectrum looks like the spectrum of a cellulose acetate.Two-dimensional NMR measurements of the perpropionylated sample did not yield more information on the structure.
Therefore it was interesting to investigate if there are sugar molecules that are incorporated in the polymer structure without being chemically bound.Diffusion measurements (DOSY) and T2 filter techniques on sample 2 were utilized to ensure that all signals detected in the NMR spectra belong to the polymer.The signals of ethanol were used as reference.Applying the T2 filter technique, the intensity of signals of small molecules with high T2 was less damped compared with the signals of the polymer (Figure 2b).This finding was underpinned by the DOSY technique (Figure 2a).Here, small molecules leave the diffusion gradient faster than the polymer and, hence, the ethanol signals loose their intensity compared with the signals of the polymer.Due to the fact that NMR studies did not gave sufficient structural information, sample 4 (DS 0.58) was subjected to methylation analysis in order to gain information on the composition of the polysaccharide derivatives studied (Table 2).The content of 1,4-linked AGUs was determined to be 33.9%,which is attributed to non-functionalized AGUs of the polymer backbone (Figure 3).The amount of 1-4,6-linked AGUs, i.e., 6-O-functionalized AGUs was 16.3%.The linkage types 1-3,4 (i.e., 3-O-glycosylated, 3.7%), 1-2,4 (i.e., 2-O-glycosylated, 3.1%), 1-3,4,6 (i.e., 3,6-di-Oglycosylated, 1.9%), 1-2,4,6 (i.e., 2,6-di-O-glycosylated, 1.6%) were found as well.It can be concluded that the reaction occurred predominantly at position 6.Reactions at positions 2 and 3 as well as multiple glycosylations were less pronounced.Terminal glucose moieties (T-Glc) can be either end groups of the polymer backbone itself or the glucose moieties attached to it.Due to the low amount of 4.9%, the content of end groups was expectedly low but also the content of glycosyl branches is low.An interesting finding was the occurrence of 34.6% of 1-2-linkages, which corresponds to a glycosidic bond at position 1 and a linkage at position 2 of a single glucose moiety.This is in agreement with the formation of a 1,2-orthoester structure, which simultaneously blocks positions 1 and 2 of the glucose molecule.
The 13 C-NMR spectrum of sample 6 synthesized in BMIMCl shows the signals of a cellulose derivative bearing acetylated glucose moieties, i.e., an additional peak for C-1' appeared at 96.7 ppm (Figure 4, top).1.5% a See Figure 3.
Interestingly, this signal is missing in the 13 C-NMR spectrum of sample 8, prepared in EMIMAc.On the contrary, the 13 C-NMR spectrum of 8 is comparable to a cellulose acetate (Figure 4, bottom).Obviously, the IL EMIMAc did not act as solvent only, but also it forced acetylation of the dissolved cellulose.This behavior has already been described in literature regarding reaction of cellulose with carboxylic acid chlorides, p-toluenesulphonic acid chloride and triphenylchloromethane. 28 Thus, reactions in BMIMCl are possible but lead to products with lower DS compared with reactions in DMA/LiCl.Moreover, use of EMIMAc as solvent is not recommended due to the predominant occurrence of acetylation instead of the orthoester formation.The products were treated with an ethanolic NaOH solution to remove the acetyl moieties in a heterogeneous procedure.The products became insoluble in DMSO but dissolved in diluted NaOH.The 1 H-NMR spectrum of sample 10 revealed complete deacetylation due to the absence of the signal at 2 ppm (CH3 of the acetyl group, Figure 5, top).The protons of the AGU were detected in the range from 3-5 ppm.The signal at 5.68 ppm was assigned to H-1 of the attached glucose residue in -configuration according to Liebert et al. 30 The 13 C-NMR spectrum of 10 is similar to that of 4 except the acetyl signals (   The peaks of the glucose moiety attached are hard to assign.However, the occurrence of some signals could be confirmed by comparison with published NMR data of 1,2-methyl orthoacetyl--D-glucopyranose: 112.9 ppm (C-8), 98.6 ppm (C-1''), 79.6 ppm (C-2''), 74.9 ppm (C-3''), 74.7 ppm (C-5''), 70.2 ppm (C-4''), 63.0 ppm (C-6''), 23.8 ppm C-7). 31These results indicated that the orthoester remained intact under the alkaline conditions applied for the deacetylation.

Conclusions
It was shown that the reaction of cellulose with 2,3,4,6-tetraacetyl-1-bromo--D-glucopyranoside in the presence of triethylamine yields derivatives bearing glucose moieties attached via 1,2orthoesters instead of a glycosidic bond, that was proved by NMR studies and methylation analysis.These derivatives are soluble in aprotic-dipolar media and at a DS of about 0.3 also in water.Water soluble derivatives are interesting products for viscosity control of aqueous media.Moreover, it is expected that this method can be used to attach other sugar moieties onto cellulose to mimic biologically active complex polysaccharides.

Experimental Section
General.The microcrystalline cellulose (Avicel®, DP 270) purchased from Sigma Aldrich (Munich, Germany) and a cellulose sample with DP 30 (obtained by acidic hydrolysis of cellulose according to Meiland et al. 32 ) were dried in vacuum over potassium hydroxide at 105°C.Lithium chloride (Sigma Aldrich, Munich, Germany) was dried in vacuum at 150°C over potassium hydroxide.The ionic liquids BMIMCl and EMIMAc were purchased from IoLiTec GmbH (Heilbronn, Germany) and used as received.Triethylamine (Sigma Aldrich, Munich, Germany) was dried over calcium hydride and distilled prior to use.All other reagents were used as received.FTIR spectra were recorded on a Nicolet AVATAR 370 DTGS spectrometer using the KBr-technique.1D-and 2D-NMR spectra were measured on a Bruker AVANCE 400 NMR spectrometer (400 MHz, Rheinstetten, Germany) with Bruker standard pulse programs and processed with MestReNova software package as well as XWINNMR.The solvents used were DMSO-d6 and CDCl3 at temperatures between 45 and 60 °C.
The glycosidic linkage analysis was determined by methylation and gas chromatographymass spectroscopy (GC-MS).Methylation of the polymers was carried out according to the procedure previously described 33 followed by GC-MS analysis of the partially methylated alditol acetates. 34,35he acetyl group content was determined according to Tanghe et al. 36 Calculation of the acetyl group content is performed according to equation 1 and the DS is calculated according to equation 2. Saponification of sample 4. Sample 4 (2.0 g) was suspended in a solution of 2.5 g potassium hydroxide and 50 mL of ethanol.The mixture was allowed to react for 24 h at 50°C under stirring in argon atmosphere.The polymer was separated by centrifugation, slurried in ethanol and neutralized with acetic acid.Impurities were removed by washing with aqueous ethanol (90:10, v/v) and the product was dried in vacuum at 60°C.Yield: 1.2 g (sample 10).H (400 MHz) 1.61 (C-7), 3.17

Figure 3 .
Figure 3. Linkage types in cellulose derivatives determined by methylation analysis.

Table 1 .
Conditions for and results of the reaction of cellulose with 3,4,6-tetraacetyl-1-bromo--D-glucopyranoside in the presence of triethylamine under homogeneous reaction conditions