<→DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN" "http://www.w3.org/TR/html4/loose.dtd"> An efficient ionic liquid supported divergent assembly of 3,6-branched glucosamine-containing pentasaccharide <→---------------------start--------------------->
  Chinese Chemical Letters  2014, Vol.25 Issue (12):1525-1530   PDF    
An efficient ionic liquid supported divergent assembly of 3,6-branched glucosamine-containing pentasaccharide
Ze-Shen Gao, Sheng Sun, Wei Li, Qing Ma, Qing Li , Zhong-Jun Li     
The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
Abstract: We utilized the glycosyl acceptor tagging method with ionic liquid support for synthesis of the core segment of Clostridium botulinum C2 toxin ligand through a divergent synthetic strategy without chromatographic purification. The total yield was 57.1% and the reaction was completed in 10 h. The efficient ionic liquid supported glycosylation and purification procedure was applied for the synthesis of branched glucosamine-containing oligosaccharides for the first time, which expanded the scope of ionic liquid supported synthesis of biologically important oligosaccharides.
Key words: Ionic liquid     Oligosaccharide     Rapid assembling    
1. Introduction

Because of the unique functionalities and structures of carbohydrates,the study of oligosaccharides has been highly valued in many areas of chemistry and biology [1]. Chemical synthesis of pure and structurally complex oligosaccharides is still a challenge due to the need of selective protection and deprotection of multiple hydroxyl groups. Moreover,traditional synthesis requires purification by chromatography after each step of glycosylation,which is not only time-consuming but also costly [2]. Solid-phase approach is one of the effective methods to improve the synthesis of oligosaccharides. It is attractive mainly because of its simple purification process,which allows convenient product isolation and automation [3, 4, 5]. In recent years,several alternative methodologies incorporating the advantages of both solid- and liquid-phase syntheses,such as polymer-supported strategy,fluorous tag method [6, 7],hydrophobically assisted switching-phase (HASP) method [8, 9, 10] and ionic liquid supported oligosaccharides synthesis (ILSOS) have been developed.

Because of their fascinating and intriguing properties,ionic liquids (ILs) have been extensively studied for their use as solvents and reaction supports [11, 12]. Several groups have successfully demonstrated the feasibility of ionic liquid supported synthesis of peptides,nucleotides,and other types of organic molecules [2, 13]. More recently,several groups utilized ionic liquid supports as phase-separation tags in the synthesis of oligosaccharides [2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23].

Synthesis of amino-containing oligosaccharides is even more challenging for the traditional liquid-phase method due to the low reactivity of amino-glycosyl donors. Special reaction conditions are often required for the complete glycosylation. Recently,synthesis of some N-linked oligosaccharides on polymer support has been reported [24]. However,there is no report on the synthesis of branched amino-oligosaccharides with ionic liquid support in literature. Given interest in the synthesis of heteroatom-containing complex oligosaccharides,we are intrigued by the possibility of assembling amino-oligosaccharides with ionic liquid support for easy purification using low-reactive amino-glycosyl donors. To demonstrate the usefulness of ionic liquid support in the synthesis of amino-oligosaccharides is important for the maturation of this strategy as a widely applicable method for preparation of biologically active oligosaccharides.

Eckhardt and co-workers reported the structure of C. botulinum C2 toxin ligand in 2000 [25]. This N-linked oligosaccharide is a GnT-V inhibitor due to its similarity in structure compared to substrates of GnT-V [26, 27]. The synthesis of segments of the Nlinked oligosaccharide 1 (Fig. 1) would be useful in determining which parts of the molecule are essential for binding. Nishizawa and co-workers synthesized some segments of 1 with the aid of ODS adsorption method based on the affinity of long alkoxybenzyl glycoside [28].

Fig. 1.The structure of Clostridium botulinum C2 toxin ligand 1.

By optimizing coupling conditions,we successfully used lowreactive amino-glycosyl donors and developed an IL supported synthesis method for the rapid assembly of 3,6-branched glucosamine-containing pentasaccharide,which is the core segment of C. botulinum C2 toxin ligand,without chromatographic purification. Furthermore,we also employed high-performance liquid chromatography (HPLC) to assess the purity of the oligosaccharide prepared by ionic liquid supported synthesis method. 2. Experimental 2.1. Standard procedure A: glycosylation using trichloro-acetimidates and TMSOTf

Acceptor and trichloroacetimidate donor were co-evaporated three times with toluene (3 × 5 mL) and dried under vacuum. After crushed 4Å molecular sieves were added,the mixture was dissolved in dry CH2Cl2 or CH2Cl2:CH3CN (10:1,only for ILsupported monosaccharide synthesis),and the solution was maintained at the desired reaction temperature. After stirring for 10 min,a solution of TMSOTf in dry CH2Cl2 was added. 2.2. Standard procedure B: purification by the ionic liquid tag method

After filtration and concentration of the reaction mixture,the residue was dissolved in CH2Cl2 and then quickly washed with saturated aq. NaHCO3 solution and brine,and dried over anhydrous Na2SO4. After evaporation in vacuo,the residue was dissolved in CH2Cl2 (2 mL/g),and 5 equiv. volume of isopropyl ether was added. The solvent was removed partially by rotary evaporation in vacuo until the residual solution was about 2 equiv. volume of the initially added CH2Cl2. The precipitate was immediately collected by centrifugation (4000 r/min,10 min). 2.3. Standard procedure C: removal of acetate protecting groups

To a solution of protected glycoside in methanol (1 g/10 mL), saturated sodium methoxide solution in methanol was added. After stirring at room temperature for 45 min,when TLC showed complete consumption of substance,the solution was neutralized with concentrated HCl and evaporated in vacuo. The residue was dissolved in CH2Cl2 and filtered. The filtrate solution was concentrated to give the target compound.

4-[(1-Methylimdazoliumhexafluorophospho)methyl]benzyl 3,6-di-O-acetyl-2,4-di-O-benzyl-a-D-mannopyranoside(4): Acceptor 2 (152 mg,0.436 mmol),donor 3 (510 mg,0.875 mmol), and promoter TMSOTf (40 µL,0.29 mmol) were used to synthesize IL supported monosaccharide 4 (281 mg,82.7%) as colorless oil. 1H NMR (400 MHz,CDCl3): δ 8.78 (s,1H,ArH),7.22-7.07 (m,36H, ArH),5.16 (s,2H,Ar-CH2-N),5.11 (dd,1H,H-3),4.82 (d,1H,H-1), 4.58-4.35 (m,6H,ArCH2),4.16 (m,2H,H-6),3.85 (t,1H,H-4),3.77 (t,1H,H-2),3.68 (s,3H,N-CH3),3.54 (m,1H,H-5),1.95 (s,3H,Ac), 1.86 (s,3H,Ac). 13C NMR (100 MHz,CDCl3): δ 170.81,170.21, 138.47,137.80,137.70,136.18,132.59,128.98,128.83,128.49, 128.39,127.88,127.77,123.78,121.97,97.22,75.70,74.89,73.77, 73.28,72.90,70.06,68.77,68.31,63.17,52.85,36.18,22.88,21.06, 20.88. HR-ESI-MS: m/z calcd. for C36H41N2O8 629.28574 [M-PF6]+, found: 629.28469.

4-[(1-Methylimdazoliumhexafluorophospho)methyl]benzyl 2- O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-[2-O-acetyl- 3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)]-2,4-di-O-benzyl- α-D-mannopyranoside (7): Acceptor 5 (80 mg,0.116mmol), donor 6 (440 mg,0.690 mmol),and promoter TMSOTf (21 μL, 0.116 mmol) were used to prepare IL supported trisaccharide 7 (142 mg,80.7%) as colorless oil. 1H NMR(400 MHz,CDCl3): δ 9.09 (s, 1H,ArH),7.34-7.19 (m,46H,ArH),5.55 (s,1H,H-2'),5.52 (s,1H,H- 2''),5.27 (s,2H,Ar-CH2-N),5.23 (s,1H,H-1),5.00 (s,1H,H-1'),4.94 (s,1H,H-1''),4.91 (t,2H,ArCH2),4.80 (d,1H,ArCH2),4.71-4.61 (m, 7H,ArCH2),4.54-4.41 (m,8H,ArCH2),4.21 (d,1H,H-6),4.08 (dd,1H, H-6),4.03-3.65 (m,19H),2.19 (s,3H,Ac),2.12 (s,3H,Ac). 13C NMR (100 MHz,CDCl3): δ 170.36,170.15,138.93,138.60,138.55,138.30, 138.26,138.01,137.89,137.85,136.57,129.05,128.85,128.50, 128.46,128.42,128.37,128.35,128.25,128.09,127.99,127.80, 127.73,127.69,127.57,123.69,121.75,99.81,98.13,96.47,78.09, 75.24,74.96,74.90,74.47,74.30,73.40,72.36,72.28,71.90, 71.59,71.37,69.22,68.92,68.84,68.48,68.38,66.45,53.57, 53.11,36.33,21.20,21.05. HR-ESI-MS: m/z calcd. for C90H97N2O18 1493.67309 [M-PF6]+,found: 1493.67758.

4-[(1-Methylimdazoliumhexafluorophospho)methyl]benzyl- 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (14): Acceptor 2 (48 mg,0.128 mmol),donor 13 (148 mg, 0.255 mmol) and promoter TMSOTf (20 μL,0.109 mmol) were used to give 14 (94 mg,90%) as colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.60 (s,1H,ArH),7.78 (s,4H,ArH),7.27-7.10 (m,6H), 5.75 (dd,1H),5.19 (s,2H,Ar-CH2-N),4.82-4.54 (m,2H,Ar-CH2), 4.33 (m,2H),4.20 (dd,1H),3.89 (m,1H),3.87 (s,3H,N-CH3),2.12 (s,3H,Ac),2.02 (s,3H,Ac),1.85(s,3H,Ac). 13C NMR (100 MHz, CDCl3): δ 170.84,170.22,169.61,138.41,136.30,134.86,132.16, 131.19,129.33,129.18,123.74,122.14,96.91,71.99,70.60,70.50, 69.07,62.06,54.61,53.55,53.12,36.38,20.89,20.72,20.54. HRESI- MS: m/z calcd. for C32H34N3O10 620.22387 [M-PF6]+,found: 620.22354.

4-[(1-Methylimdazoliumhexafluorophospho)methyl]benzyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucosyl-(1→2)- 3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-[3,4,6-tri-O-acetyl- 2-deoxy-2-phthalimido-β-D-glucosyl-(1→2)-3,4,6-tri-O-benzyl- α-D-mannopyranosyl-(1→3)]-2,4-di-O-benzyl-α-D-mannopyranoside (15): Acceptor 11 (51 mg,0.0328 mmol),donor 13 (133 mg,0.229 mmol) and promoter TMSOTf (6 μL,0.0328 mmol) were used to prepare IL supported pentasaccharide 15 (64 mg, 87.3%) as colorless oil. 1H NMR (400 MHz,CDCl3): δ 9.42 (s,1H, ArH),7.90-6.99 (m,54H,ArH),5.81-3.22 (m,55H),2.89-1.81 (m, 21H). 13C NMR (100 MHz,CDCl3): δ 170.61,170.46,170.26,170.09, 170.00,169.76,169.55,169.42,169.25,167.64,138.63,138.60, 138.54,138.36,138.27,138.13,138.03,137.92,137.77,137.50, 137.31,137.18,134.48,134.33,134.23,134.17,134.13,132.30, 132.22,132.10,131.94,131.82,131.70,131.48,131.39,131.25, 131.18,131.03,130.86,130.37,129.91,129.25,129.16,129.09, 128.98,128.53,128.33,128.20,128.08,128.02,127.91,127.81, 127.70,127.63,127.59,127.53,127.42,127.37,127.30,127.19, 126.99,126.66,126.45,126.34,126.29,126.10,124.19,124.06, 123.95,123.78,123.55,123.50,123.39,123.30,121.64,121.59, 121.49,121.42,120.63,99.57,99.04,97.18,96.63,96.03,75.13, 74.57,74.25,73.37,72.84,72.60,71.92,71.03,70.55,70.53,70.49, 70.45,70.11,69.63,69.06,68.74,68.50,67.04,65.73,62.23,62.05, 61.80,61.68,61.56,61.11,60.98,54.99,54.65,54.38,54.36,54.30, 54.11,53.50,53.13,36.47,36.41,36.34,36.29,35.70,29.76,29.48, 29.33. HR-ESI-MS: m/z calcd. for C126H131N4O34 2243.86392 [MPF6]+,found: 2243.86281. 3. Results and discussion 3.1. Assembly of 3,6-branched trimannoside

We started with the glycosylation of modified ionic liquid support 2 as previously synthesized [22] with excess of trichloroacetimidate 3 in CH2Cl2/CH3CN,which was carried out using TMSOTf (50 mol%) at 0 ℃ for complete transformation (Scheme 1) with standard procedure A. Standard purification B procedure for ionic liquid supported synthesis produced almost pure monosaccharide 4 in 82.7% yield with complete α-selectivity.

Scheme 1.Ionic liquid supported rapid synthesis of trimannoside. Reagents and conditions: (a) 2 equiv. donor 3,0.5 equiv. TMSOTf,0 ℃,30 min; (b) NaOMe in MeOH,60 min; (c) 6 equiv. donor 6,1 equiv. TMSOTf,0 ℃,30 min.

The glycosyl acceptor 5 was prepared by treatment of glycoside 4 with NaOMe in MeOH with standard procedure C. The reaction of diol 5 with trichloroacetimidate 6 catalyzed by TMSOTf afforded trisaccharide 7 in 80.7% yield after quick purification by the ionic liquid tag phase separation (procedure A and B) (Scheme 1). 3.2. Optimization of glycosylation conditions

Ionic iquid supported synthesis requires high conversion rate of coupling reaction. However,amino-glycosyl donors are relatively inactive. We used the glycosylation of N-Troc-trichloracetimidate 8 with ionic liquid supported monosaccharide 9 as a model reaction to optimize the reaction conditions for lowreactive amino-sugar donors (Table 1 and Scheme 2). The reaction condition was quite harsh and the yield was not good.

Table 1
Optimization of glycosylation conditions with N-Troc.

Scheme 2.The model reaction of N-Troc protected amino donor 8.

The trisaccharide 7 was converted to diol 11 by treatment with NaOMe in MeOH. As discussed above,the coupling conditions were selected as follows: TMSOTf 100 mol%,reflux (35 ℃),2 h,repeated twice. Therefore,the synthesis of the core glucosamine-containing segment 12 was successfully achieved in the yield 68% by the glycosylation of 8 with 11 (Scheme 3).

Scheme 3.Synthesis of the pentasaccharide. Reagents and conditions: 6 equiv. donor 8 (2 equiv. for the second glycosylation); 0.5 equiv. TMSOTf repeated for two times for 2 h each under reflux.

Due to the low yield of 12 in 68%,the double glycosylation,and long reaction time,we turned to evaluate other amino protecting groups which could allow mild and high yield of glycosylation.N-Phth group was a classic amino protecting group used for preparing the β-glucoside via the neighboring effect. The glycosylation of N-Phth-trichloracetimidate 13 with ionic liquid acceptor 2 was performed as a new model reaction. The yield of 14 was higher and the reaction only needed to be carried out once (Scheme 4).

Scheme 4.The model reaction of N-Phth protected amino donor 13 with ionic liquid 2. 2 equiv. donor,TMSOTf 50 mol%,0 ℃,30 min,90%.
3.3. Assembly of the core glucosamine-contained pentasaccharide

As discussed above,the coupling conditions were selected as follows: TMSOTf 100 mol%,0 ℃,2 h. Therefore,the synthesis of the core glucosamine-containing segment 15 was successfully achieved by the glycosylation of 11 with 13 in the yield of 87.3% (Scheme 5).

Scheme 5.Synthesis of the pentasaccharide with ionic liquid support. Reagents and conditions: 7 equiv. donor 13; 1 equiv. TMSOTf; 0 ℃,2 h.
3.4. HPLC analysis of oligosaccharides synthesized with ionic liquid support

The structures of 4,7,12,14 and 15 were supported by 1H NMR and 13C NMRspectra and further confirmed by HR-ESI-MS analysis. Galan and co-workers used HPLC to analyze the oligosaccharides prepared by using ionic liquid support [21]. A HPLC method was also used to analyze the purity of our sample.Wefound that,due to the unique properties of ionic liquid species,common mobile phases were not suitable for their analysis. We used a solution of KPF6 in acetonitrile as the mobile phase,and sharper peaks were generated. As shown in Fig. 2,the glycosylation and purification cycle gave product 4 in 98% purity.

Fig. 2.The model reaction of N-Troc protected amino donor 8.

As shown in Fig. 3,the glycosylation and purification cycle gave product 7 in 80% purity. The desired product 7 (4.6 min) was the major product and excess trichloroacetimidate 6 was efficiently removed. The difficulty in purification of the branched oligosaccharides may result in lower purity from 98% to 80%.

Fig. 3.HPLC chromatogram of prepared trisaccharide 7 following ionic liquid supported purification. Mobile phase A: acetonitrile,10 mmol/L KPF6 added,B: H2O. Flow rate: 1 mL/min,100% A/0% B,UV absorbance at 210 nm.

HPLC analysis of the final reaction products after rapid purification showed one major peak with 75% purity,which is shown in Fig. 4.

Fig. 4.HPLC chromatogram of pentasaccharide 14 following ionic liquid supported purification. Mobile phase A: acetonitrile,10 mmol/L KPF6 added,B: H2O. Flow rate: 0.5 mL/min,80% A/20% B,UV absorbance at 210 nm.

Details of each step through the whole assembling pathway are listed in Table 2. And segment 15 was prepared in 57.1% overall yield within 10 h.

Table 2
Cycles used for IL supported assembly of glucosamine-contained pentamannoside.
4. Conclusions

In summary,we investigated the glycosyl acceptor tagging strategy with ionic liquid for the synthesis of glucosaminecontaining oligosaccharides. By optimizing coupling conditions, we found an efficient glycosylation-purification procedure using low-reactive amino-sugar donor with ionic liquid supported synthesis,which expanded the scope of ionic liquid supported synthesis of oligosaccharides. With this method,the core segment of C. botulinum C2 toxin ligand was synthesized. This work demonstrated the usefulness of the present procedure for the construction of branched glucosamine-containing oligosaccharide in a divergent synthetic strategy without chromatographic purification. We also developed an efficient HPLC method for the analysis of reaction products. Our method for the efficient synthesis of oligosaccharides on ionic liquid support could be used to produce oligosaccharides on a large scale.


This research was supported by the National Basic Research Program of China (973 Program,No. 2012CB822100),the National Key Technology R&D Program ‘‘New Drug Innovation’’ of China (No. 2012ZX09502001-001) and the National Natural Science Foundation of China (Nos. 91213301,21232002).

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