Synthesis of Cyclic β-1,6-Oligosaccharides by Electrochemical Polyglycosylation of glucosamine monomers

Synthesis of protected precursors of cyclic β -1,6-oligoglucosamines by electrochemical polyglycosylation of thioglycosides as a monomer is performed. The monomer with 2,3 -oxazolidinone protecting group afforded the cyclic disaccharide exclusively. Cyclic o ligosaccharides up to trisaccharide were obtained using the monomer with 2-deoxy-2-azido group.

cyclic oligosaccharide; electrochemical glycosylation; glucosamine; polyglycosylation 2

Electrochemical polymerization of organic molecules is an important process to prepare functional materials such as conducting polymers [1-5]. Electrochemical reactions can be controlled by electric potential or current, electrodes, and electrolytes, which are not available in conventional chemical reactions. Therefore, electrochemical polymerizations can be utilized selective synthesis . Cyclic oligosaccharides are important class of host molecules and some natural cyclic oligosaccharides are produced by enzymatic processes; however, their chemical syntheses are still primitive [6-10]. We have been interested in preparation of cyclic oligosaccharides under electrochemical conditions and electrochemical conversion of linear oligosaccharides of glucosamine in to the corresponding cyclic oligosaccharides by intramolecular glycosylation (Figure 1 a) [ 11]. One -pot two-step synthesis via electrochemical polyglycosylation and intramolecular glycosylation has also been achieved to synthesize unnatural cyclic oligosaccharides of glucosamine (Figure 1b) [12]. Here, we report direct synthesis of cyclic oligoglucosamines via electrochemical polymerization of thioglycoside monomers which are derived from glucosamine hydrochloride. 3 Figure 1. Preparation of cyclic oligoglucosamines. (a) via intramolecular glycosylation. (b) via polyglycosylation and intramolecular glycosylation. 4

and Discussion Electrochemical Polyglycosylation of 2 -deoxy-2-phtalimide thioglycoside monomer We initiated our research from the electrochemical polyglycosylation of monomers 6 with 2-deoxy-2-phtalimide (2-PhthN) group (Table 1). The monomer 6a (R3 = R4 = Bz) was completely consumed with the slight excess amount of total charge (Q = 1.05 F/mol); however, 1,6-anhydrosugar 7a (R3 = R4 = Bz) was formed as a major product together with cyclic disaccharide 8a (R3 = R4 = Bz) (entry 1). The monosaccharide 6b (R3 = Ac, R4 = Bn) was also completely consumed under the same reaction conditions; however, the yield of 1,6-anhydrosugar 7b (R3 = Ac, R4 = Bn) was lower than that of 7a (entry 2). Because no linear oligosaccharides were obtained, we reduced the amount of total charge from 1.05 to 0.525 F/mol (entry 3). Linear disaccharides 9b (R3 = Ac, R4 = Bn) and trisaccharide 10b (R3 = Ac, R4 = Bn) were obtained in 13% and 6% yields, respectively. The protecting group R3 of 3-OH was changed from acetyl (Ac) group to benzyl (Bn) group; however, conversion and yields of linear oligosaccharides 9c and 10c were decreased and the corresponding cyclic disaccharide 8c was not obtained at all (entry 4). In all cases the major product was 1,6-anhydrosugar 7 which was the product of intramolecular glycosylation of monomer 6. The proposed mechanism is shown in Figure 2. Anodic oxidation of thioglycoside 6 generated radical cation 11 which is converted to glycosyl triflate 12. 1,6-Anhydrosugar 7 is produced via the 4C1 to 1C4 conformational change of the pyran ring to generate cation intermediate 13. Therefore, prevention of the conformational change might be necessary to synthesize larger cyclic oligosaccharides. 5 Table 1. Electrochemical polyglycosylation of monomers 6 with 2-PhthN group. entry R3 R4 total charge Q (F/mol) conv. yield of 7 yields of oligosaccharides 8 9 10 1 Bz Bz 1.05 >99% 73% 7a 3% 8a - - 2 Ac Bn 1.05 >99% 28% 7b 6% 8b - - 3 Ac Bn 0.525 67% 25% 7b 7% 8b 13% 9b 6% 10b 4 Bn Bn 0.525 59% 25% 7c - 4% 9c 2% 10c Figure 2. Proposed reaction mechanism of formation of 1,6-anhydrosugar 7. Electrochemical Polyglycosylation of 2,3-oxazolidione thioglycoside monomer To avoid formation of 1,6 -anhydrosugar we introduced N-acetyl-2,3-oxazolidione protecting group into the thioglycoside monomer 14 (Figure 3) [13,14]. The electrochemical polyglycosylation of 14 was carried out in the presence of 2,6-di-tert- 6 butyl-4-methylpyridine (DTBMP) to ensure the formation of β-glycosidic bonds [ 15]. Although we could suppress formation of 1,6-anhydrosugar 15, cyclic disaccharide 16 was obtained as an exclusive product. The optimized structure of 15 calculated by DFT (B3LYP/6-31G(d)) suggested that the pyran ring preferred the boat conformation because the chair conformation of the pyran ring was controlled by the introduction of the 2,3-oxazolidinone protecting group (See Supporting Information for DFT calculation). Therefore, it was proved that the 2,3-oxazolidinone protecting group was powerful enough to prevent intramolecular glycosylation of monomer 14; however, it was not useful to prevent intramolecular glycosylation of the linear disaccharide and promote the formation of larger cyclic oligosaccharides. Figure 3 . Electrochemical p olyglycosylation of monomer 14 with 2,3 -oxazolidione protecting group. Electrochemical Polyglycosylation of 2-deoxy-2-azido thioglycoside monomer Based on the results of table 1 and figure 3 , we changed the substituent of C -2 position of the thioglycoside monomer from phthalimide (PhthN) group to azido (N 3) group which has no neighboring group effect. Although glycosyl donors with N 3 group at C-2 position have been used for α-selective glycosylation [16,17], we have already found that β-selective glycosylation proceeded using a glycosyl donor with N 3 group under the electrochemical conditions [18]. The results of electrochemical 7 polyglycosylation using the thioglycoside monomer 17 with N3 group are summarized in Table 2. Cyclic trisaccharide 19a was obtained together with cyclic disaccharide 18a and the trace amount of linear and cyclic tetrasaccharides by the introduction of N 3 group (entry 1). Cyclic disaccharide 18b and linear trisaccharide 20b were produced with monomer 17b with 3,4-di-O-benzyl group (entry 2). Although the protecting group (R3) at 3-OH also affected the product distribution, formation of the corresponding 1,6- anhydrosugars were not observed in both cases . NMR data suggested that c yclic trisaccharide 19a contains one α-glycosidic bond and two β-glycosidic bonds. Based on these results we assume that the formation of α-glycosidic bond is crucial to produce cyclic trisaccharide 19a (Figure 4). Moreover, the α-glycosidic bond might be formed in the first step and linear disaccharide 21α which did not afford cyclic disaccharide should be produced as an intermediate of 19a. Table 2. Electrochemical polyglycosylation of monomer 17 with 2-azido group. entry R3 conv. yields of oligosaccharides 18 19 20 1 Ac >99% 49% 18a 16% 19a - 2 Bn 73% 14% 18b - 13% 20b 8 Figure 4. Proposed reaction mechanism of formation of cyclic trisaccharide 19a.

In conclusion , we have investigated synthesis of cyclic β-1,6-oligoglucosamines under the electrochemical polyglycosylation condition. The choice of protecting group of monomers is important to prevent intramolecular glycosylation which forms 1,6 - anhydrosugar as a side product. It was revealed that the formatio n of cyclic disaccharide must be controlled to produce cyclic β-1,6-trisaccharide. Further optimization of monomers and another synthetic approach using dimers for production of larger cyclic oligosaccharides are in progress in our laboratory. Experimental Electrochemical polyglycosylation (Figure 3) has been performed using our second - generation automated electrochemical synthesizer equipped with the H -type divided electrolysis cell. Thioglycoside 14 (0.40 mmol, 186 mg), Bu4NOTf (1.0 mmol, 393 mg), DTBMP (2.0 mmol, 411 mg), and dry CH 2Cl2 (10 mL) were added to the anodic chamber. Triflic acid (0.4 mmol, 35 μL) and CH2Cl2 (10 mL) were added to the cathodic chamber. Electrolysis was performed at -20 °C under the constant current condition 9 until 1.2 F/mol of total charge was consumed. Then the reaction temperature was elevated to 0 °C and the temperature was kept for 1 h. The reaction was quenched with Et3N (0.5 mL), and the reaction mixture was dissolved in EtOAc and washed with water to remove electrolyte. It was further washed with aqueous 1 M HCl solution and dried over Na 2SO4. Then the solvent was removed under reduced pressure and the crude product (220 mg) was purified with preparative GPC to obtain pure cyclic oligosaccharides 16 (0.125 mmol, 79.7 mg, 62%). Supporting Information Supporting Information File 1: File Name: SI-Cyclic oligoglucosamine File Format: PDF Title: Supporting Information of Synthesis of Cyclic β -1,6-Oligosaccharides by Electrochemical Polyglycosylation of glucosamine monomers

The authors acknowledge the financial support by the Grant -in-Aid for Scientific Research (JP23H01961). The contents of this paper have been published by Md Azadur Rahman as a PhD thesis at Tottori University in 2023.

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