{"paper_id":"1cb98119-3e68-4efa-b587-4f85daa71a73","body_text":"License and Terms: This document is copyright 2024 the Author(s); licensee Beilstein-Institut.\nThis is an open access work under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0). Please note that the reuse,\nredistribution and reproduction in particular requires that the author(s) and source are credited and that individual graphics may be subject to special legal provisions.\nThe license is subject to the Beilstein Archives terms and conditions: https://www.beilstein-archives.org/xiv/terms.\nThe definitive version of this work can be found at https://doi.org/10.3762/bxiv.2024.23.v1\nThis open access document is posted as a preprint in the Beilstein Archives at https://doi.org/10.3762/bxiv.2024.23.v1 and is\nconsidered to be an early communication for feedback before peer review. Before citing this document, please check if a final,\npeer-reviewed version has been published.\nThis document is not formatted, has not undergone copyediting or typesetting, and may contain errors, unsubstantiated scientific\nclaims or preliminary data.\nPreprint Title Synthesis of Cyclic β-1,6-Oligosaccharides by Electrochemical\nPolyglycosylation of glucosamine monomers\nAuthors Md Azadur Rahman, Hirofumi Endo, Takashi Yamamoto, Shoma\nOkushiba, Norihiko Sasaki and Toshiki Nokami\nPublication Date 16 Apr. 2024\nArticle Type Full Research Paper\nSupporting Information File 1 SI-Cyclic oligoglucosamine.pdf;  9.2 MB\nORCID® iDs Toshiki Nokami - https://orcid.org/0000-0001-5447-4533\n\n1 \nSynthesis of Cyclic β-1,6-Oligosaccharides by Electrochemical \nPolyglycosylation of glucosamine monomers \nMd Azadur Rahman 1, Hirofumi Endo 1, Takashi Yamamoto1, Shoma Okushiba 1, \nNorihiko Sasaki1,2, and Toshiki Nokami*1,2 \n \nAddress: 1Department of Chemistry and Biotechnology, Tottori University , 4-101 \nKoyamacho-minami, Tottori city, 680 -8552 Tottori, Japan,  2Center for Research on \nGreen Sustainable Chemistry, Faculty of Engineering, Tottori University, 4 -101 \nKoyamacho-minami, Tottori city, 680-8552 Tottori, Japan \n \nEmail: Toshiki Nokami – tnokami@tottori-u.ac.jp \n* Corresponding author \nAbstract \nSynthesis of protected precursors of cyclic β -1,6-oligoglucosamines by \nelectrochemical polyglycosylation of thioglycosides as a monomer is performed. The \nmonomer with 2,3 -oxazolidinone protecting group afforded the cyclic disaccharide \nexclusively. Cyclic o ligosaccharides up to trisaccharide were obtained using the \nmonomer with 2-deoxy-2-azido group. \nKeywords \ncyclic oligosaccharide; electrochemical glycosylation; glucosamine; polyglycosylation \n\n2 \nIntroduction \nElectrochemical polymerization of organic molecules is an important process to \nprepare functional materials such as conducting polymers  [1-5]. Electrochemical \nreactions can be controlled by electric potential or current, electrodes, and electrolytes, \nwhich are not available in conventional chemical reactions. Therefore, electrochemical \npolymerizations can be utilized selective synthesis . Cyclic oligosaccharides are \nimportant class of host molecules and some natural cyclic oligosaccharides are \nproduced by enzymatic processes; however, their chemical syntheses are still primitive \n[6-10]. We have been interested in preparation of cyclic oligosaccharides under \nelectrochemical conditions and electrochemical conversion of linear oligosaccharides \nof glucosamine in to the corresponding cyclic oligosaccharides  by intramolecular \nglycosylation (Figure 1 a) [ 11]. One -pot two-step synthesis via electrochemical \npolyglycosylation and intramolecular glycosylation  has also been achieved to \nsynthesize unnatural cyclic oligosaccharides of glucosamine (Figure 1b) [12]. Here, we \nreport direct synthesis of cyclic oligoglucosamines via electrochemical polymerization \nof thioglycoside monomers which are derived from glucosamine hydrochloride. \n \n\n3 \n \nFigure 1. Preparation of cyclic oligoglucosamines. (a) via intramolecular glycosylation. \n(b) via polyglycosylation and intramolecular glycosylation. \n  \n\n4 \nResults and Discussion \nElectrochemical Polyglycosylation of 2 -deoxy-2-phtalimide \nthioglycoside monomer \nWe initiated our research from the electrochemical polyglycosylation of monomers \n6 with 2-deoxy-2-phtalimide (2-PhthN) group (Table 1). The monomer 6a (R3 = R4 = \nBz) was completely consumed with the slight excess amount of total charge (Q = 1.05 \nF/mol); however, 1,6-anhydrosugar 7a (R3 = R4 = Bz) was formed as a major product \ntogether with cyclic disaccharide 8a (R3 = R4 = Bz) (entry 1). The monosaccharide 6b \n(R3 = Ac, R4 = Bn) was also completely consumed under the same reaction conditions; \nhowever, the yield of 1,6-anhydrosugar 7b (R3 = Ac, R4 = Bn) was lower than that of \n7a (entry 2). Because no linear oligosaccharides were obtained, we reduced the \namount of total charge from 1.05 to 0.525 F/mol (entry 3). Linear disaccharides 9b (R3 \n= Ac, R4 = Bn) and trisaccharide 10b (R3 = Ac, R4 = Bn) were obtained in 13% and 6% \nyields, respectively. The protecting group  R3 of 3-OH was changed from acetyl (Ac) \ngroup to benzyl (Bn) group; however, conversion and yields of linear oligosaccharides \n9c and 10c were decreased and the corresponding cyclic disaccharide 8c was not \nobtained at all (entry 4). In all cases the major product was 1,6-anhydrosugar 7 which \nwas the product of intramolecular glycosylation of monomer 6. The proposed \nmechanism is shown in Figure 2. Anodic oxidation of thioglycoside 6 generated radical \ncation 11 which is converted to glycosyl triflate 12. 1,6-Anhydrosugar 7 is produced via \nthe 4C1 to 1C4 conformational change of the pyran ring to generate cation intermediate \n13. Therefore, prevention of the conformational change might be necessary to \nsynthesize larger cyclic oligosaccharides. \n \n\n5 \nTable 1. Electrochemical polyglycosylation of monomers 6 with 2-PhthN group. \n \nentry R3 R4 total charge \nQ (F/mol) conv. yield of 7 yields of oligosaccharides \n8 9 10 \n1 Bz Bz 1.05 >99% 73% 7a 3% 8a  - - \n2 Ac Bn 1.05 >99% 28% 7b 6% 8b  - - \n3 Ac Bn 0.525 67% 25% 7b 7% 8b  13% 9b  6% 10b  \n4 Bn Bn 0.525 59% 25% 7c - 4% 9c  2% 10c  \n \n \nFigure 2. Proposed reaction mechanism of formation of 1,6-anhydrosugar 7. \n \nElectrochemical Polyglycosylation of 2,3-oxazolidione thioglycoside \nmonomer \nTo avoid formation of 1,6 -anhydrosugar we introduced N-acetyl-2,3-oxazolidione \nprotecting group into the thioglycoside monomer 14 (Figure 3)  [13,14]. The \nelectrochemical polyglycosylation of 14 was carried out in the presence of 2,6-di-tert-\n\n6 \nbutyl-4-methylpyridine (DTBMP) to ensure the formation of β-glycosidic bonds [ 15]. \nAlthough we could suppress formation of 1,6-anhydrosugar 15, cyclic disaccharide 16 \nwas obtained as an exclusive product. The optimized structure of 15 calculated by DFT \n(B3LYP/6-31G(d)) suggested that the pyran ring preferred the boat conformation \nbecause the chair conformation of the pyran ring was controlled by the introduction of \nthe 2,3-oxazolidinone protecting group (See Supporting Information for DFT \ncalculation). Therefore, it was proved that the 2,3-oxazolidinone protecting group was \npowerful enough to prevent intramolecular glycosylation of monomer 14; however, it \nwas not useful  to prevent intramolecular glycosylation of the linear disaccharide and \npromote the formation of larger cyclic oligosaccharides. \n \nFigure 3 . Electrochemical p olyglycosylation of monomer 14 with 2,3 -oxazolidione \nprotecting group. \n \nElectrochemical Polyglycosylation of 2-deoxy-2-azido thioglycoside \nmonomer \nBased on the results of table 1 and figure 3 , we changed the substituent of C -2 \nposition of the thioglycoside monomer from phthalimide (PhthN) group to azido (N 3) \ngroup which has no neighboring group effect. Although glycosyl donors with N 3 group \nat C-2 position have been used for α-selective glycosylation [16,17], we have already \nfound that β-selective glycosylation proceeded using a glycosyl donor with N 3 group \nunder the electrochemical conditions  [18]. The results of electrochemical \n\n7 \npolyglycosylation using the thioglycoside monomer 17 with N3 group are summarized \nin Table 2. Cyclic trisaccharide 19a was obtained together with cyclic disaccharide 18a \nand the trace amount of linear and cyclic tetrasaccharides by the introduction of N 3 \ngroup (entry 1). Cyclic disaccharide 18b and linear trisaccharide 20b were produced \nwith monomer 17b with 3,4-di-O-benzyl group (entry 2). Although the protecting group \n(R3) at 3-OH also affected the product distribution, formation of the corresponding 1,6-\nanhydrosugars were not observed  in both cases . NMR data suggested that c yclic \ntrisaccharide 19a contains one α-glycosidic bond and two β-glycosidic bonds. Based \non these results we assume that the formation of α-glycosidic bond is crucial to produce \ncyclic trisaccharide 19a (Figure 4). Moreover, the α-glycosidic bond might be formed \nin the first step and linear disaccharide 21α which did not afford cyclic disaccharide \nshould be produced as an intermediate of 19a. \n \nTable 2. Electrochemical polyglycosylation of monomer 17 with 2-azido group. \n \nentry R3 conv. yields of oligosaccharides \n18 19 20 \n1 Ac >99% 49% 18a 16% 19a - \n2 Bn 73% 14% 18b - 13% 20b \n \n\n8 \n \nFigure 4. Proposed reaction mechanism of formation of cyclic trisaccharide 19a. \nConclusion \nIn conclusion , we have investigated synthesis of cyclic β-1,6-oligoglucosamines \nunder the electrochemical polyglycosylation condition. The choice of protecting group \nof monomers is important to prevent intramolecular glycosylation which forms 1,6 -\nanhydrosugar as a side product. It was revealed that the formatio n of cyclic \ndisaccharide must be controlled to produce cyclic β-1,6-trisaccharide. Further \noptimization of monomers and another synthetic approach using dimers for production \nof larger cyclic oligosaccharides are in progress in our laboratory. \nExperimental \nElectrochemical polyglycosylation (Figure 3) has been performed using our second -\ngeneration automated electrochemical synthesizer equipped with the H -type divided \nelectrolysis cell. Thioglycoside 14 (0.40 mmol, 186 mg), Bu4NOTf (1.0 mmol, 393 mg), \nDTBMP (2.0 mmol, 411 mg), and dry CH 2Cl2 (10 mL) were added to the anodic \nchamber. Triflic acid (0.4 mmol, 35 μL) and CH2Cl2 (10 mL) were added to the cathodic \nchamber. Electrolysis was performed at -20 °C under the constant current condition \n\n9 \nuntil 1.2 F/mol of total charge was consumed. Then the reaction  temperature was \nelevated to 0 °C and the temperature was kept for 1 h. The reaction was quenched \nwith Et3N (0.5 mL), and the reaction mixture was dissolved in EtOAc and washed with \nwater to remove electrolyte. It was further washed with aqueous 1 M HCl solution and \ndried over Na 2SO4. Then the solvent was removed under reduced pressure  and the \ncrude product (220 mg) was purified with  preparative GPC to obtain pure  cyclic \noligosaccharides 16 (0.125 mmol, 79.7 mg, 62%). \nSupporting Information  \nSupporting Information File 1: \nFile Name: SI-Cyclic oligoglucosamine \nFile Format: PDF \nTitle: Supporting Information of Synthesis of Cyclic β -1,6-Oligosaccharides by \nElectrochemical Polyglycosylation of glucosamine monomers \nAcknowledgements \nThe authors acknowledge the financial support by the Grant -in-Aid for Scientific \nResearch (JP23H01961).  The contents of this  paper have been published by Md \nAzadur Rahman as a PhD thesis at Tottori University in 2023. \nReferences \n1. Daiaz, A. F.; Kanazawa, K. K.; Gardini, G. P. J. Chem. Soc., Chem. Commun. 1979, \n14, 635–636. doi: 10.1039/c5py01407g \n2. Tanaka, K.; Shichiri, T.; Wang, S.; Yamabe, T. Synth. Met. 1988, 24, 203–215. doi: \n10.1016/0379-6779(88)90258-5 \n\n10 \n3. 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