Combinatorial Synthesis of Carbohydrate Cluster on Tree-Type Linker with Orthogonally Cleavable Parts

Abstract

We have successfully developed a synthetic strategy for the combinatorial solid-phase synthesis of carbohydrate clusters on a tree-type linker with two types of orthogonally cleavable parts. The combination of three types of spacers, two types of carbohydrate ligands, and two types of orthogonal cleavage provided a 12-member carbohydrate cluster library (6 dimers and 6 tetramers).

Key words

combinatorial synthesis - solid-phase synthesis - ortho­gonal cleavage - carbohydrate cluster - library - tree-type linker

References

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9

Alcohol 3 was prepared from glycerol over 6 steps: 1. pivaloylation of two 1°-hydroxyl groups; 2. protection of the remained hydroxyl group with THP group; 3. alkaline hydrolysis; 4. di-mesylation; 5. di-azidation; 6. acid-catalyzed hydrolysis.

11

Commercially available from Argonaut Technologies, San Carlos, California: http://www.argotech.com.

15

The characteristic IR absorption was observed at 2092, 2056, 2018 cm^-1.

16

The cleavage product was filtered through the reverse phase C-18 column (Varian Bond Elut C18, 500 mg). Spectral data of 9: ¹H NMR (400 MHz, D₂O, 30 °C): δ = 2.78 (dd, 4 H, J = 6.3, 6.3 Hz), 3.20 (dd, 2 H, J = 7.2, 14.0 Hz), 3.27-3.33 (m, 2 H), 3.30 (s, 4 H), 3.45 (dd, 2 H, J = 7.7, 9.7 Hz), 3.57 (dd, 2 H, J = 3.4, 9.7 Hz), 3.58-3.62 (m, 2 H), 3.65-3.71 (m, 4 H), 3.79 (dt, 2 H, J = 6.3, 10.6 Hz), 3.80 (m, 1 H), 3.85 (brd, 2 H, J = 3.4 Hz), 4.01 (dt, 2 H, J = 6.3, 10.6 Hz), 4.34 (d, 2 H, J = 7.7 Hz, anomeric). ¹³C NMR (67.8 MHz, D₂O, 30 °C): δ = 34.2, 37.8, 45.5, 63.6, 71.0, 71.3, 71.5, 73.4, 75.4, 77.9, 105.6, 165.2, 175.6. MS (ESI-TOF): 651 [M + H]⁺.

17

The cleavage product was filtered through the reverse phase C-18 column (Varian Bond Elut C18, 500 mg). Spectral data of 10: ¹H NMR (400 MHz, D₂O, 30 °C): δ = 2.56 (br s, 4 H), 2.85 (dd, 4 H, J = 5.3, 6.3 Hz), 3.33 (dd, 4 H, J = 6.3, 14.0 Hz), 3.36 (s, 8 H), 3.43 (dd, 4 H, J = 4.4, 14.0 Hz), 3.50 (dd, 4 H, J = 7.7, 9.7 Hz), 3.61-3.68 (m, 8 H), 3.70-3.80 (m, 8 H), 3.82-3.88 (m, 6 H), 3.90 (br d, 4 H, J = 2.9 Hz), 3.97 (quintet, 1 H, J = 3.9 Hz), 4.07 (dt, 4 H, J = 6.3, 10.6 Hz), 4.31 (br s, 4 H), 4.40 (d, 4 H, J = 7.7 Hz, anomeric). ¹³C NMR (67.8 MHz, D₂O, 30 °C): δ = 28.1, 34.8, 43.1, 60.1, 63.6, 70.6, 71.2, 71.3, 71.4, 73.3, 73.4, 75.5, 77.9, 78.0, 80.8, 86.6, 105.6, 175.5. MS (ESI-TOF): 1455.5 [M + Na]⁺.

18

All products were confirmed by ESI-TOF mass spectra and the complete assignments of chemical shifts (¹H NMR) based on ¹H-¹H COSY spectra.

19

FITC-ManBSA was prepared by coupling of Fluorescein isothiocyanate (FITC) with coupled with mannosylated bovine serum albumine (ManBSA) in carbonate buffer (pH 9.5) at r.t. Purification of FITC-ManBSA was achieved by purified by gel-filteration on Sephedex G-25 using phosphate buffered saline as an eluent.

20

Dimer-CM and tetramer-CM could not be examined because of their less solubility in water.

21

FITC-ManBSA derived mean fluorescence intensity of PEMs was analyzed cytofluorimetrically by gating viable CD₄ ⁺ population. The values are means ± SD from three separate experiments.