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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Org Chem. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2888776
NIHMSID: NIHMS134365

Imidazolium Cation Supported Solution-phase Assembly of Homo-linear α(1→6)–Linked Octamannoside – An Efficient Alternate Approach for Oligosaccharide Synthesis

Abstract

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An efficient, simple convergent assembly of a homo-linear α(1→6)-linked octamannosyl thioglycoside was obtained starting from imidazolium cation–tagged mannosyl fluoride and thiomannoside using block couplings. During chain elongation glycosylation reactions no column chromatographic purifications were used.

The significance of carbohydrates is now well valued as these play pivotal roles in several key biological systems and are essential parts of every cell surface that is crucial in cell to cell recognition and communication.1 Access to pure well defined oligosaccharide and glycoconjugate structures needed as biochemical tools is still a challenge to the chemists. However, the synthesis of these are made possible by controlled stereo- and regio-specific, chemoselective, orthogonal, and bi-directional glycosylation reactions via solution-phase as well as polymer supported solution- or solid-phase organic synthesis.2,3 Although highly successful, solid-phase synthesis suffers from a series of drawbacks due to heterogeneous reaction conditions and stepwise characterization. Alternate solution-phase polymer supported methods allow efficient homogeneous conditions, but issues with loading capacity and product losses during purification are still challenging. Fluorous chemistry using a fluorous biphasic system and fluorous tag for fluorous separations are also recently exploited for the synthesis of oligosaccharides.4

Access to newer, straightforward and efficient methods to produce biologically relevant oligosaccharides is still warranted and the use of imidazolium type cationic species (ionic liquids, ILs)5 as support during glycosylation processes can be approached to facilitate and condense the process of oligosaccharide synthesis. Imidazolium cationic species are reported to be used as anchors for reaction substrates in the synthesis of peptides,6 nucleotides7 and other types of organic molecules.5,8 IL–linkers are real molecules and have defined structures and molecular weights in contrast to polymer linkers, which are functionalized materials with variable loading capabilities. Recently, we and others have successfully utilized imidazolium cations as phase separation tags on glycosyl donors in glycosylation reactions.9,10 The main feature of IL-supported substrates are their solubility properties, as solubility can be altered towards particular types of solvents by simply varying the anions. Thereafter, IL–supported species can be purified from the reaction mixture by simply washing the product with a solvent in which the IL–tagged compounds are not soluble. Whenever required, the IL support can be clipped off and purified by a simple phase separation technique. In principle, synthesis of almost pure oligosaccharides can be achieved efficiently with minimal column chromatographic purification via this approach in homogeneous reaction conditions. In addition, IL–tagged substrates are easily characterized by conventional spectroscopic techniques. A mannosyl fluoride donor linked with imidazolium hexafluorophosphoacetyl was previously used by us and iterative coupling11 with thioglycoside acceptors led to the solution–phase synthesis of a linear α(1→6)–linked tetramannoside in an efficient manner.9

The physical properties of imidazolium cation–tagged protected sugars beyond tetrasaccharide are not clear and we sought to further examine the solubility properties of the growing chain as well as their phase-tag purification by simple washing with solvents. To test the limits of a IL–tag assisted approach to oligosaccharide synthesis, access to a model homo–linear α(1→6)–linked octamannosyl thioglycoside 1 on imidazolium cation–tagged substrates using chemoselective/orthogonal glycosylation was chosen (Figure-1). d-Mannose oligomers are profoundly present in nature and are essential substructures in many biologically important glycoconjugates such as N-glycans, fungal and bacterial cell wall mannans and GPI anchors.12 Branched oligomannan syntheses are reported in the literature using several different methodologies13 including solution- and solid-phase polymer supports14 and fluorous-tag15 assisted solution-phase. Specifically, the homo-linear α(1→6) oligomannans were previously synthesized in solution-phase16,17 and by automated solid-phase18 approach. In a tedious conventional solution-phase process, we recently synthesized a linear α(1→6)– octamannosyl fluorescent probe,17 via the two-stage activation procedure for the synthesis of oligosaccharides,19 combining the chemistry of thioglycosides with that of glycosyl fluorides.

Figure 1
Retrosynthetic analysis of IL-supported synthesis of α(1→6) octamannoside using imidazolium cation–tagged donors.

With our interest in developing newer simple and efficient approaches to synthesize oligosaccharides in small to large quantities, we report herein, a convergent synthesis of octasaccharide 1 by means of conveniently accessible IL–tagged p-thiotolyl mannoside 2 and IL–tagged mannosyl fluoride 3 and block glycosylation. The IL–tagged mannosyl synthons 2 and 3 were synthesized from a common known p-thiotolyl mannoside 4.9,17 The thioglycoside 4 was chosen as the starting precursor because it can be prepared in a multigram scale in just two steps from D-mannose through a known precursor 1,6-di-O-acetyl-2,3,5-tri-O-benzoyl-α-D-mannose.20 The thioglycoside 4 was converted to IL-tagged glycosyl fluoride 3 using DAST and NBS at −20 °C followed by installation of imidazolium tag as reported in excellent yield.9

IL-tagged thioglycoside 2 was synthesized in large scale from 4 in three steps as shown in Scheme-1. First, the selective removal of the acetyl group at C-6 in compounds 4 was induced by an AcCl/MeOH/CH2Cl2 mixture (0.1:20:20 v/v, 12.0 mL/mmol) and afforded 6-OH glycosides 5 in 93% yield.9 Glycoside 5 was then treated with chloroacetyl chloride to 6-O-chloroacetylated product 6. Lastly, glycoside 6 was treated overnight with N-methylimidazole under reflux conditions in CH3CN and subsequent exchange of chloride anion with hexafluorophosphino anion (PF6) was obtained in the same pot by the reaction with KPF6. The reaction was cooled to room temperature and removal of solvent afforded a solid, which was then suspended in CHCl3 and filtered. The filtrates were concentrated to a solid and washed several times with diethyl ether to produce almost pure IL–tagged thioglycoside 2 in large scale, equipped with a robust participating benzoyl group at C2 to ensure α–selectivity and a removable temporary imidazolium cation–tagged O–acetyl protecting group at the C6, for further chemical manipulations.

Scheme 1
Synthesis of IL-tagged thiomannopyranoside donor 2

A detailed IL–supported synthesis of target octasaccharide 1 is illustrated in Scheme 2. With large quantities of IL–tagged p-tolylthio mannoside 2 and mannosyl fluoride 3 available, we assembled IL-tagged disaccharides 8 and 9. Glycosylation of IL-tagged mannosyl fluoride donor 3 with thiomannoside acceptor 5 using coupling reagents21 Cp2HfCl2 and AgClO4 in CH2Cl2 progressed as expected to give almost pure IL–tagged thiodisaccharide 8 after washing the product by diethyl ether and the product was analytically compared with the previously prepared sample by us.9 On the other hand, glycosylation of IL–tagged thiomannosyl donor 2 with fluoride acceptor 7 was carried out in presence of promoter NIS and triflic acid. The workup and simple washing purification protocol with diethyl ether produced almost pure IL–tagged dimannosyl fluoride 9 in 80% yield. The 1H and 13C NMR, and ESIMS spectral studies of 9 supported the structure and the reducing end anomeric carbon was observed as a doublet at δ 105.39 (JC-F = 223.3 Hz) in the 13C NMR spectrum.

Scheme 2
Synthesis of α(1→6)-linked octamannan 1.

In the present process, the IL–tagged glycosylated products, after purification by simple washing, were split in to two portions. In the subsequent glycosylation reaction, one portion was used as a glycosyl donor and the other portion was utilized as a glycosyl acceptor after removing the IL–tag and purification by bi-phasic separation technique. In comparison to our previous iterative glycosylation approach11 for the IL-supported synthesis of linear tetramannoside in which the monosaccharide block was added one at a time, this block glycosylation efficiently produced the target octasaccharide in fewer steps.

Now, to proceed further, each IL–tagged disaccharides 8 and 9 was divided into two portions. On one portion of each disaccharide, the IL–tag was conveniently detached by stirring with aqueous saturated NaHCO3 in a H2O/ether (1:1) biphasic solvent mixture in the presence of a phase transfer catalyst Bu4N+I. Concentration of the ether layer provided almost pure acceptor disaccharides 10 and 11 as shown by their 1H and 13C NMR spectra, and were further used as such in glycosylation reactions. With the acceptor thiodisaccharide 10 and glycosyl fluoride 11 in hand, we next executed the [2+2] block glycosylation reaction and accessed IL–supported α(1→6)–tetramannose thioglycoside 12 and α(1→6)–tetramannosyl fluoride 13. In a glycosylation process similar for the synthesis of disaccharides 8 and 9, the IL–tagged thioglycoside 8 was reacted with mannosyl fluoride acceptor 11 and IL–tagged mannosyl fluoride 9 was reacted with thioglycoside acceptor 11 in presence of coupling promoters NIS–TfOH and Cp2HfCl2–AgClO4 respectively. The workup and simple washing purification protocol with diethyl ether produced almost pure IL–tagged tetrasaccharides 12 and 13 for next step usage.

Again, a portion of synthesized IL–tagged thiomannosyl tetrasaccharide 12 was treated with an aqueous saturated NaHCO3 in a H2O/ether (1:1) biphasic solvent mixture in the presence of Bu4N+I. After concentration of the ether layer almost pure acceptor thiomannosyl tetrasaccharide 14 was obtained and further utilized in glycosylation reaction without purification. To facilitate the synthesis of IL-tagged α(1→6)–linked octamannosyl thioglycoside 15, a convergent block coupling of [4+4] was commenced with the overnight coupling reaction of donor IL–tagged mannosyl fluoride 13 and acceptor thiomannoside 14 in CH2Cl2 using coupling reagents Cp2HfCl2 and AgClO4. The usual workup and simple washing purification protocol with diethyl ether produced almost pure IL–tagged octasaccharides 15. Finally, the IL support from the octasaccharide 15 was removed, as described for 14, and resulted in almost pure homo–linear α(1→6)–linked octamannosyl thioglycoside 1 in 83% yield. The structure of 1 was supported by its 1H and 13C NMR spectra and the final structural confirmation was obtained by ESIMS analysis of 1, which showed a peak at 3942.8 [M+Na]+ corresponding to the molecular formula C223H184O64SNa.

In conclusion, we demonstrate a successful, orthogonal block synthesis of an octasaccharide on an imidazolium cation support with minimal column chromatographic purification as an alternative to existing oligosaccharide synthetic approaches on support materials. The IL–tagged thioglycosides and IL-tagged glycosyl fluorides were chemoselectively activated in presence of each other by coupling reagents NIS–TfOH and Cp2HfCl2–AgClO4 respectively. All intermediate products were analyzed by 1H and 13C NMR, and MS spectral techniques. Further development of IL-supported solution-phase techniques will provide carbohydrate chemists a new tool to achieve their synthetic goals in an efficient and cost-effective approach and will broaden the diversity of targets for glycobiology.

Experimental Section

General procedure for coupling with IL-tagged thioglycoside

IL supported donor thioglycoside (1.0 eq), acceptor fluoride glycoside (1.2 eq) and activated powdered 4Å molecular sieves in CH2Cl2 were cooled at -20 °C under argon atmosphere. The mixture was stirred for 10 min, and NIS (1.2 eq) followed by TfOH (0.1 eq) were added to initiate coupling. The reaction mixture was allowed to stir for 30 min and the reaction was quenched by the addition of saturated aqueous NaHCO3. It was diluted with CHCl3 and the organic phase was filtered through a celite pad. The filtrate was washed with 10% Na2S2O3 (10 mL) and the organic layer was dried over anhydrous Na2SO4. It was concentrated under vacuum to a solid which was purified by repeated washing with diethyl ether and the solvent was removed by decantation. The solid residue was then dried under vacuum to afford IL supported fluoride saccharide.

General procedure for coupling with IL-tagged fluoride donor

The IL-tagged fluoride (1.0 eq) and the acceptor thioglycoside (1.1 eq) were dissolved in CH2Cl2 under Ar atmosphere in presence of activated 4Å molecular sieves. The reaction mixture was cooled to 0 °C and coupling reagents AgClO4 (1.5 eq) and Cp2HfCl2 (1.1 eq) were added. The reaction mixture was stirred overnight, filtered and concentrated under vacuum. The residue was washed with diethyl ether several times and the solvent was removed by decantation. The residue was finally dried under vacuum to give pure IL-tagged thioglycoside.

p-Tolyl 2,3,4-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-D-mannopyranosyl-2,3,4-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-1-thio-2,3,4-tri-O-benzoyl-α-D-mannopyranoside (1)

IL-tagged octaasaccharide 15 (500 mg, 0.12 mmol) was dissolved in bi-phasic solvent mixture diethyl ether–water (15 mL, 1:1 v/v). To it were added saturated aqueous NaHCO3 solution (10 mL) and Bu4N+I- (70 mg). The reaction mixture was stirred for 8 h at room temperature; the ether layer was separated and washed with brine (2×10 mL). The ether layer was dried over anhydrous Na2SO4, concentrated and the residue was dried under high vacuum to produce octasaccharide 1 as a colorless solid (388 mg, 83% yield). m.p. 110 °C. Rf = 0.2 (2:1 Cyclohexane–EtOAc). 1H NMR (300 MHz, CDCl3): δ 8.20 (m, 12H), 8.08 (m, 20H), 7.88 (m, 18H), 7.54 (m, 30H), 7.43 (m, 22H), 7.28 (m, 34H), 7.14 (d, 2H, J = 8.0 Hz), 6.42 (t, 1H, J = 10.2 Hz), 6.22 (m, 5H), 6.10 (m, 4H), 6.00 (m, 7H), 5.90 (m, 4H), 5.74 (m, 4H), 5.23 (s, 1H), 5.14 (d, 1H, J = 8.3 Hz), 5.05 (s, 1H), 4.97 (m, 5H), 4.81 (s, 1H), 4.38 (dd, 1H, J = 3.9, 11.4 Hz), 4.18 (m, 7H), 3.96 (m, 2H), 3.81 (m, 5H), 3.65 (d, 1H, J = 8.3 Hz), 3.55 (d, 1H, J = 10.1 Hz), 3.35 (m, 7H), 2.22 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 166.9, 165.8, 165.7, 165.5, 165.5, 165.3, 165.2, 138.5, 133.8, 133.6, 133.4, 133.2, 133.1, 132.9, 130.2, 130.0, 129.8, 129.5, 129.5, 129.4, 129.3, 129.1, 128.9, 128.8, 128.6, 128.43, 98.4, 98.2, 97.9, 86.9, 72.1, 71.1, 70.9, 70.6, 70.2, 69.7, 69.6, 69.3, 67.2, 66.9, 66.6, 66.5, 66.3, 65.9, 65.8, 60.8, 21.2. ESIMS: m/z 3942.8737 [M+Na]+ calcd for C223H184O64SNa, found 3942.7.

Supplementary Material

1_si_001

Acknowledgments

This work was made possible by grant number R21AI059270 from National Institute of Health and Seed grant from Western Illinois University Research Council.

Footnotes

Supporting Information Available: Complete experimental procedures and characterization (1H and 13C NMR spectra). This material is available free of charge via the internet at http://pubs.acs.org.

References

1. a) Varki A. Glycobiology. 1993;3:97–130. [PubMed] b) Dwek RA. Chem Rev. 1996;96:683–720. [PubMed] c) Sears P, Wong CH. Cell Mol Life Sci. 1998;54:223–152. [PubMed] d) Rudd PM, Elliot T, Cresswell P, Wilson IA, Dwek RA. Science. 2001;291:2370–2376. [PubMed]
2. a) Levy DE, Fugedi P. The Organic Chemistry of Sugars. CRC Press; Noca Raton, FL: 2006. b) Hanessian S. Preparative Carbohydrate Chemistry. Marcel Dekker; New York: 1996. c) Demchenko AV. Handbook of Chemical Glycosylation: Advances in Stereoselectivity and Therapeutic Relevance. Wiley-VCH; Weinheim: 2008.
3. a) Seeberger PH. Chem Soc Rev. 2008;37:19–28. [PubMed] b) Seeberger PH. Solid Support oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries. John Wiley; New York: 2001. and refrences therein.
4. Zhang W. Chem Rev. 2009;109:749–795. [PubMed]
5. a) Murugesana S, Linhardt RJ. Curr Org Synth. 2005;2:437–451. b) Miao W, Chan TH. Acc Chem Res. 2006;39:897–908. [PubMed] c) El Seoud OA, Koschella A, Fidale LC, Dorn S, Heinze T. Biomacromolecules. 2007;8:2629–2647. [PubMed] d) Martin MAP, Frizzo CP, Moreira DN, Zanatta N, Bonacorso HG. Chem Rev. 2008;108:2015–2050. [PubMed] e) Borikar SP, Daniel T, Paul V. Tetrahedron Letters. 2009;50:1007–1009. f) Wang J, Song G, Peng Y, Zhu Y. Tetrahedron Letters. 2008;49:6518–6520. g) Galan MC, Brunet C, Fuebsanta M. Tetrahedron Letters. 2009;50:442–445.
6. a) Miao W, Chan TH. J Org Chem. 2005;70:3251–3255. [PubMed] b) He X, Chan TH. Org Lett. 2007;9:2681–2684. [PubMed]
7. Donga RA, Khaliq-Uz-Zaman SM, Chan TH, Damha MJ. J Org Chem. 2006;71:7901–7910.
8. a) Legeay JC, Vanden Eynde JJ, Bazureau JP. Tetrahedron. 2008;64:5328–5335. b) Grzyb JA, Batey RA. Tetrahedron Letters. 2008;49:5279–5282.
9. Pathak AK, Yerneni CK, Young Z, Pathak V. Org Lett. 2008;10:145–148. [PMC free article] [PubMed]
10. a) Huang JY, Lei M, Wang YG. Tetrahedron Lett. 2006;47:3047–3050. b) He X, Chan TH. Synthesis. 2006:1645–1651.
11. In this process, after each coupling reaction between IL–tagged mannosyl fluoride donor and thiomannosyl acceptor, the IL–tag was removed from the glycosylated product to obtain thiomannoside acceptor. In the next glycosylation step, this thiomannoside acceptor was further coupled with another equivalent of IL–tagged mannosyl fluoride donor and chain elongation was achieved.
12. Few selected articles. a) Kobayashi H, Mitobe H, Takahashi K, Yamamoto T, Shibata N, Suzuki S. Arch Biochem Biophys. 1992;294:662–669. [PubMed] b) Mandal DK, Bhattacharya L, Koenig SH, Brown RD, Oscarson S, Brewer CF. Biochemistry. 1994;33:1157–1162. [PubMed] c) Brennan PJ. Annu Rev Biochem. 1995;64:29–63. [PubMed] d) Chatterjee D, Khoo KH. Glycobiology. 1998;8:113–120. [PubMed] e) Hadar F, Daniel AM, Kurt D, William IW. Science. 2001;294:2163–2166. [PubMed] f) Carole AB, Shigeki K, Hamachi I. J Mol Biol. 2002;322:881–889. [PubMed] g) Botos I, O'Keefe BR, Shenoy SR, Cartner LK, Ratner DM, Seeberger PH, Boyd MR, Wlodawer A. J Biol Chem. 2002;277:34336–34342. [PubMed]
13. Few selected articles. a) Heng L, Ning J, Kong F. J Carbohydr Chem. 2001;20:285–296. b) Zhu Y, Chen L, Kong F. Carbohydr Res. 2002;337:207–215. [PubMed] c) Ning J, Heng L, Kong F. Tetrahedron Lett. 2002;43:673–675. d) Xing Y, Ning J. Tetrahedron Assym. 2003;14:1275–1283. e) Ratner DM, Plante OJ, Seeberger PH. Eur J Org Chem. 2002:826–833. f) Crich D, Banerjee A, Yao Q. J Am Chem Soc. 2004;126:14930–14934. [PubMed] g) Lo'pez JC, Agocs A, Uriel C, Go'meza AM, Fraser-Reid B. Chem Commun. 2005:5088–5090. h) Jayaprakash KN, Chaudhuri SR, Murty CV, Fraser-Reid B. J Org Chem. 2007;72:5534–5545. [PubMed]
14. a) Douglas SP, Whitfield DM, Krepinsky JJ. J Am Chem Soc. 1995;117:2116–2117. b) Andrade RB, Plante OJ, Melean LG, Seeberger PH. Org Lett. 1999;1:1811–1814. [PubMed]
15. Jaipuri FA, Pohl NL. Org Biomol Chem. 2008;6:2686–2691. [PubMed]
16. Zhu Y, Kong F. Carbohydr Res. 2001;332:1–21. [PubMed]
17. Aqueel MS, Pathak V, Pathak AK. Tetrahedron Letters. 2008;49:7157–7160. [PMC free article] [PubMed]
18. Liu X, Wada R, Boonyarattanakalin S, Castagner B, Seeberger PH. Chem Commun. 2008:3510–3512. [PubMed]
19. Nicolaou KC, Ueno H. In: Preparative Carbohydrate Chemistry. Hanessian S, editor. Marcel Dekker; New York: 1996. pp. 313–338. and references therein.
20. Heng L, Ning J, Kong F. Carbohydr Res. 2001;331:431–437. [PubMed]
21. Suzuki K, Maeta H, Matsumoto T, Tsuchihashi GI. Tetrahedron Lett. 1988;29:3571–3574.