Oligosaccharide Synthesis by Affinity Separation Based on Molecular Recognition between Podand Ether and Ammonium Ion

Abstract

We previously reported a new synthetic methodology termed ‘synthesis based on affinity separation’ (SAS) in which the desired tagged compound was separated from the reaction mixture by solid-phase extraction using specific molecular recognition of the tag. The interaction between a crown ether tag and polymer-­supported ammonium ion was initially employed for SAS. In the present study, a new SAS method using a podand-type tag, a ­pseudo-benzo-31-crown-10 structure, was elaborated for oligosaccharide synthesis. The podand tag was much easier to synthesize than the corresponding crown ether. The podand moiety was ­attached to the monosaccharide residue via appropriate linkers. ­After glycosylation of the tagged monosaccharide with a glycosyl donor, the reaction mixture was subjected to the affinity separation. The desired compounds possessing the podand tag were effectively separated by the affinity between the podand and the ammonium ion. Continuous flow synthesis by integration of a microreactor and the present SAS system was applied for high throughput oligo­saccharide synthesis.

oligosaccharide synthesis - affinity separation - high throughput synthesis - linker - glycosylation

References

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6

The podand tag with a shorter chain length showed weaker affinity to the ammonium ion column, whereas the tag having longer chain length, such as 6, showed comparable affinity with the corresponding crown ether derivatives.

7

The maximum adsorption of 8 to ArgoPore-NH₃ ⁺·CF₃COO^- was determined as follows: the solid-phase extraction column (Varian, Bond Elut empty cartridges with frits, 6 mL capacity) was filled with ArgoPore^®-NH₂ (1.74 g, Argonout Technologies, Inc). The resin column was washed with CH₂Cl₂-MeOH (1:1) and CH₂Cl₂ (or toluene). The amino groups on the resins were changed to ammonium ions with 10% TFA in CH₂Cl₂ (or toluene) and then excess TFA was washed with CH₂Cl₂ (or toluene). A sufficient quantity of the sample in CH₂Cl₂ (or toluene) was charged to the ArgoPore-NH₃ ⁺·CF₃COO^- column and then the column was washed with the same solvent to remove the excess sample. Compound 8 adsorbed in the column was then eluted with CH₂Cl₂-MeOH (1:1). The maximum adsorption of 8 was determined to be 138 mg (6.9% to amino groups) in toluene and 58.4 mg (2.9% to amino groups) in CH₂Cl₂.

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13

Benzylcarbonyl and benzyl-type tags can be removed by catalytic hydrogenolysis using Pd catalyst.

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17

Glycosylation with nitropyridyl glycoside gave better results than with glycosyl fluoride and thioglycoside. The fucosylation stopped at 50% consumption of 33. The reaction mixture was applied to SAS to give a mixture of 33 and trisaccharide 35. After TFA was removed by liquid-liquid separation, the mixture was subjected to glycosylation with fucosyl donor 34, again. This procedure was repeated twice and the resulting compound 35 was finally purified by silica gel chromatography.

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IMM micromixer: http://www.imm-main2.de/.

22

Representative Procedure of SAS Method. Preparation of 32.
To a solution of the acceptor 30 (77 mg, 76 µmol), trichloroacetimidate 31 (112 mg, 227 µmol), and molecular sieves 4 Å in CH₂Cl₂ (1.0 mL) was added TMSOTf (2.7 µL, 15 µmol) at 0 °C under Ar atmosphere. After the reaction mixture was stirred for 1 h at r.t., the resulting mixture was filtered to remove the molecular sieves before applying to the affinity separation. The mixture was directly charged onto ArgoPore-NH₃ ⁺·CF₃COO^- filled in a syringe-like column (Varian, Bond Elut empty cartridges with frits, column size: 2.0 cm × 8.5 cm, resin 3.8 g), prepared according to ref. 7. After untagged compounds were washed off with CH₂Cl₂ (200 mL), the tagged product 32 was eluted with CH₂Cl₂-MeOH (1:1, 50 mL). Evaporation of the solvents afforded the desired product 32 as a yellow oil (95 mg, 93%): ESI-MS(positive): m/z = 1367.50 [M + Na]⁺. ¹H NMR (500 MHz, CDCl₃): δ = 7.32 (d, J = 1.5 Hz, 1 H, ClAzb), 7.24 (d, J = 8.2 Hz, 1 H, ClAzb), 7.12 (d, J = 8.2 Hz, 1 H, ClAzb), 6.53 (d, J = 2.2 Hz, 2 H, -C₆H₃-CH₂-OCO), 6.47 (t, J = 2.1 Hz, 1 H, -C₆H₃-CH₂-OCO), 5.96-5.80 (m, 2 H, -CH₂-CH=CH₂ × 2), 5.31-5.16 (m, 6 H, H-4′, -CH₂-CH=CH₂ × 2, H-2′), 5.09 (s, 2 H, -C₆H₃-CH₂-OCO), 4.95 (dd, J = 3.4, 10.4 Hz, 1 H, H-3′), 4.90 (d, J = 11.6 Hz, 1 H, ClAzPh-CH₂), 4.81 (d, J = 3.7 Hz, 1 H, H-1), 4.78 (d, J = 10.0 Hz, 1 H, NH), 4.60 (d, J = 7.9 Hz, 1 H, H-1′), 4.56-4.48 (m, 4 H, ClAzPh-CH₂, OCO-CH₂-CH=CH₂, H-6a), 4.23 (dd, J = 3.7, 11.5 Hz, 1 H, H-6b), 4.15-4.08 [m, 5 H, -C₆H₃ (OCH₂)₂, -CH₂-CH=CH₂], 3.98-3.93 (m, 2 H, -CH₂-CH=CH₂, H-2), 3.89-3.53 {m, 33 H, -C₆H₃[OCH₂CH₂-(OCH₂CH₂)₂-OCH₂CH₂OMe]₂, H-4, H-5, H-5′, H-6′, H-3}, 3.37 (s, 6 H, MeO × 2), 2.13, 2.05, 1.96 (s, 12 H, Ac × 4).