Synthesis of rhamnogalacturonan I oligosaccharides: Synthesis of a tetrasaccharide intermediate

Article abstract of DOI:10.1016/S0957-4166(98)00494-7

A hydroxyl protected tetrasaccharide intermediate, corresponding to a segment of the rhamnogalacturonan I polysaccharide, has been synthesized using the glycosyl imidate technique. This tetrasaccharide is designed to allow for further elongation and branching.

Full text of DOI:10.1016/S0957-4166(98)00494-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 10 (1999) 17–20
Synthesis of rhamnogalacturonan I oligosaccharides: synthesis of
a tetrasaccharide intermediate
Jamie R. Rich, Robert S. McGavin, Rebecca Gardner and Kerry B. Reimer ∗
Department of Chemistry, University of Northern British Columbia, 3333 University Way, Prince George, British Columbia,
Canada, V2N 4Z9
Received 4 November 1998; accepted 1 December 1998
Abstract
A hydroxyl protected tetrasaccharide intermediate, corresponding to a segment of the rhamnogalacturonan I
polysaccharide, has been synthesized using the glycosyl imidate technique. This tetrasaccharide is designed to
allow for further elongation and branching. © 1999 Elsevier Science Ltd. All rights reserved.
The study of the molecular mechanisms of plant glycobiology is a new and expanding field. It is
becoming clear that the role of complex carbohydrates in plants goes beyond energy storage mol-
ecules or structural elements.1–4 Rhamnogalacturonan I (RG-I) is a complex polysaccharide found in
plant tissues. The backbone of RG-I is composed of an [–α-D-galactopyranosyluronic acid-(1-2)-α-L-
rhamnopyranosyl-(1-4)–] repeating unit. The 4-position of the rhamnosyl residues are substituted with
side chains containing L-arabinose, D-galactose, L-fucose and D-glucuronic acid residues in linear and
branched configurations.5 Monoclonal antibodies are being used as molecular probes to investigate
the function of this and other plant polysaccharides in plant growth and development.6,7 The results
of these types of studies are meaningful only if the binding specificities of the antibodies in use are
fully understood. Oligosaccharides of known structure and high purity are invaluable, when used in
competitive binding assays, in characterizing the specificity of an antibody binding site. It is difficult
to obtain pure samples of oligosaccharides in sufficient quantities for such studies, hence the need to
prepare such structures by chemical synthesis. This paper describes the overall strategy for the synthesis
of branched RG-I oligosaccharides, and the synthesis of a tetrasaccharide intermediate corresponding to
a segment of the RG-I polysaccharide backbone (Fig. 1).
Tetrasaccharide 1 is a key intermediate in our overall synthetic strategy to synthesize RG-I oligo-
saccharides. The protecting group pattern of compound 1 is designed so that the 2-O-acetyl group of
the terminal rhamnosyl unit may be selectively removed. This allows for chain extension by addition
of disaccharide 21. Alternatively, removal of the 4-O-allyl groups of the rhamnosyl residues allows for
addition of further glycosyl units to give branched oligosaccharides. Finally, removal of the blocking
∗
Corresponding author. E-mail: reimerk@unbc.ca
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00494-7

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J. R. Rich et al. / Tetrahedron: Asymmetry 10 (1999) 17–20
Figure 1.
groups, and oxidation8 of the primary hydroxyls of the galactosyl residues introduces the carboxylic acid
groups found in the native RG-I polysaccharide. This strategy allows the oligosaccharide to be built up
using the more stable galactose as a temporary substitute for galacturonic acid.
The rhamnosyl donor 6 was prepared from the ethyl thioglycoside 29 in a series of reactions that
was performed without purification between steps (Scheme 1). Compound 2 was treated with trimethyl
orthoacetate in dimethylformamide and a catalytic amount of p-toluenesulfonic acid. The resulting
orthoester 3 was then treated with allyl bromide in dimethylformamide to give 4. The orthoester of 4
was then opened under acidic conditions to give the 2-O-acetyl derivative 5. The hydroxyl group of 5
was then benzoylated to give the fully blocked rhamnosyl donor 6.
Scheme 1. Reagents and conditions: (a) trimethyl orthoacetate, PTSA, in DMF; (b) allyl bromide, NaH, in DMF; (c) 80%
aqueous acetic acid; (d) benzoyl chloride in pyridine, 6 from 2 69.8% yield; (e) α,α-dimethoxytoluene, PTSA, in DMF, 80°C,
8 from 7 79.3% yield, 13 from 12 45% yield; (f) benzyl bromide, NaH, in DMF: (g) HCl in MeOH (0.5 mL of a 1.0 M soln),
in CH2Cl2; (h) benzoyl chloride, NaOH (1.25 M)/CH2Cl2, [CH3(CH2)3]4N(HSO4), 11 from 8 70.1% yield, 16 from 13 60.3%
yield
Galactosyl acceptor 11 was prepared starting with methyl β-D-galactopyranoside 7. The 2- and 3-
positions were protected as benzyl ethers by treatment of the 4,6-benzylidene derivative 8 with benzyl
bromide and sodium hydride in dimethylformamide to give 9. The benzylidene group was removed
and the resulting diol 10 was selectively benzoylated under phase transfer conditions10 to give the 6-
O-benzoyl derivative 11. The galactosyl donor 16 was prepared in a similar series of reactions starting
from tert-butyl β-D-galactopyranoside 1211 (prepared from tetra-O-acetyl-D-galactopyranosyl bromide
and tert-butanol).
Reaction of 11 with 6 in dichloromethane with N-iodosuccinimide and a catalytic amount of triflic
acid12,13 gave the disaccharide 17 (Scheme 2). Similarly, reaction of 16 with 6 gave disaccharide 19.
Selective removal of the 2-O-acetyl group of 17 was achieved by treatment with methanolic HCl14 to

J. R. Rich et al. / Tetrahedron: Asymmetry 10 (1999) 17–20
19
give disaccharide 18. The 1H-NMR spectrum of 18 showed the loss of the signal due to the acetyl group,
and the H-2 of the rhamnosyl residue had shifted to 4.34 ppm for compound 18, from 5.61 ppm for
compound 17, consistent with deacetylation of the 20-position. Disaccharide 19 was transformed into
a glycosyl donor by treatment of a dichloromethane solution of 19 with trichloroacetic acid to give a
mixture of hemiacetals 20,15,16 which where then reacted with trichloroacetonitrile and DBU17 to give
the trichloroacetimidate derivative 21. Disaccharides 18 and 21 were then reacted together with catalytic
amounts of trimethylsilyl triflate18 to give the tetrasaccharide 1.19 The newly formed glycosidic linkage
between the galactosyl and the rhamnosyl residues of 1 was formed in the desired α-configuration. This
determination was made based on the 13C–1H coupling constant for C-1 of the internal galactosyl unit.
This coupling constant was measured from a 1H-detected 13C–1H correlation spectrum of 1 and found to
be 159 Hz; a value characteristic of α-glycosidic linkages. One-dimensional and two-dimensional NMR
spectra20 for all products were consistent with the proposed structures.
Scheme 2. Reagents and conditions: (a) N-iodosuccinimide, triflic acid (cat.), in CH2Cl2; (b) methanolic HCl in CH2Cl2, 58.5%
yield; (c) trichloroacetic acid, in CH2Cl2, 62%; (d) trichloroacetonitrile, DBU, in CH2Cl2; (e) trimethylsilyl triflate in CH2Cl2
In a test reaction, using disaccharide 17 as a model compound for tetrasaccharide 1, the 4-O-allyl
group of 17 was successfully removed. This result indicates that selective deprotection of the 4-positions
of tetrasaccharide 1 should be possible, and hence generation of branched oligosaccharides. Tetrasac-
charide 1 represents a key synthetic intermediate in the synthesis of branched chain oligosaccharides
corresponding to sequences found in the RG-I polysaccharide.
Acknowledgements
We wish to thank the Natural Sciences and Engineering Research Council of Canada for financial
support of this work.
References
1. Cote, F.; Hahn, M. G. Plant Molecular Biology 1994, 26, 1379–1411.
2. Fry, S. C. Biochemical Society Symposium 1994, 60, 5–14.
3. Darvill, A.; Bergmann, C.; Cervone, F.; Delorenzo, G.; Ham, K. S.; Spiro, M. D.; York, W. S.; Albersheim, P. Biochemical
Society Symposium 1994, 60, 89–94.
4. Albersheim, P.; An, J. H.; Freshour, G.; Fuller, M. S.; Guillen, R.; Ham, K. S.; Hahn, M. G.; Huang, J.; Oneill, M.;
Whitcombe, A.; Williams, M. V.; York, W. S.; Darvill, A. Biochemical Society Transactions 1994, 22, 374–378.

20
J. R. Rich et al. / Tetrahedron: Asymmetry 10 (1999) 17–20
5. McNeil, M.; Darvill, A. G.; Albersheim, P. Plant Physiol. 1982, 78, 1586–1591.
6. Freshour, G.; Clay, R. P.; Fuller, M. S.; Albersheim, P.; Darvill, A. G.; Hahn, M. G. Plant Physiol. 1996, 110, 1413–1429.
7. Balestrini, R.; Hahn, M. G.; Faccio, A.; Mendgen, K.; Bonfante, P. Plant Physiol. 1996, 111, 203–213.
8. Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1990, 205, 147–159.
9. Auzanneau, F.-I.; Bundle, D. R. Carohydr. Res. 199, 212, 13–24.
10. Garegg, P. J.; Kvarnstrom, I.; Niklasson, A.; Niklasson, G.; Svensson, S. C. T. J. Carbohydr. Chem. 1993, 12, 933–953.
11. Cocker, D.; Jukes, L. E.; Sinnott, M. L. J. Chem. Soc., Perkin Trans. 2 1973, 190–194.
12. Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron Lett. 1990, 31, 1331–1334.
13. Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31, 4313–4316.
14. Byramova, N. E.; Ovchinnikov, M. V.; Backinowsky, L. V.; Kochetkov, N. K. Carbohydr. Res. 1983, 124, C8–C11.
15. Westman, J.; Nilsson, M. J. Carbohydr. Chem. 1995, 14, 949–960.
16. Banaszek, A. Carbohydr. Res. 1998, 306, 379–385.
17. Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl. 1980, 19, 732–735.
18. Wegman, B.; Schmidt, R. R. J. Carbohydr. Chem. 1987, 6, 357–375.
19. The yield for this reaction is not optimised. The main product of the reaction on TLC corresponded to the isolated
tetrasaccharide; however, the reaction was carried out on relatively small scale (33 mg of 18) and losses were incurred
during work-up and chromatography.
1
20. H (300.13 MHz) NMR data for selected compounds. 1: Chemical shifts for 1H and 13C were measured from a
COSY spectrum and a 1H-detected 13C–1H correlation spectrum, respectively. 8.15–7.10 (40H, aromatic), 5.77 (2H,
OCH2CH_CH2 for the two allyl groups), 5.66 (H-2), 5.60 (H-3), 5.54 (H-3), 5.29 (H-1), 5.06 (H-1), 4.81 and 4.50
(ABq, OCH2Ph), 4.79 and 4.61 (ABq, OCH2Ph), 4.78 and 4. 62 (ABq, OCH2Ph), 4.63 (H-6a), 4.56 (H-6b), 4.53 (H-1),
4.51 and 4.38 (ABq, OCH2Ph), 4.42 (H-2), 4.32 (H-1), 4.13 (H-4), 4.03 (H-5), 4.00 (H-5), 3.96 (H-4), 3.95 (H-6a), 3.89
(H-5), 3.88 (H-3 and H-6b), 3.83 (H-2), 3.80 (H-5), 3.75 (H-2), 3.60 (3H, OCH3), 3.61 (H-4), 3.52 (H-3), 3.46 (H-4),
1.98 (3H, OCOCH3), 1.37 (3H, H3-6), 1.19 (3H, H3-6); 13C (75.47 MHz) 105.23 (J
159 Hz, C-1), 99.81 (J
13
1
13
1
C−
H
C−
H
13
1
13
1
173 Hz, C-1), 98.39 (J
168 Hz, C-1), 94.78 (J
169 Hz, C-1). 18: 8.07 (m, 4H, aromatic), 7.63–7.20 (m,
C−
H
C−
H
16H, aromatic), 5.80 (m, 1H, OCH2CH_CH2), 5.46 (dd, 1H, J3 ,2 3.0 Hz, J3 ,4 7.0 Hz, H-30), 5.19 and 5.08 (m, 2H,
OCH2CH_CH2), 5.17 (1H, H-10), 4.93 and 4.74 (ABq, 2H, JA,B 11.0 Hz, OCH2Ph), 4.79 and 4.61 (ABq, 2H, JA,B 11.5
Hz, OCH2Ph), 4.59 (dd, 1H, J6b,5 6.5 Hz, J6a,6b 11.0 Hz, H-6a), 4.51 (dd, 1H, J6b,5 5.9 Hz, H-6b), 4.34 (m, 1H, H-20), 4.30
0
0
0 0
(d, 1H, J1,2 7.5 Hz, H-1), 4.23 and 4.11 (m, 2H, OCH2CH_CH2), 4.05 (m, 1H, H-4), 3.97 (dq, 1H, J5 ,4 9.0 Hz, H-50),
3.77 (m, 2H, H-5 and H-2), 3.58 (s, 3H, OCH3), 3.54 (dd, 1H, J3,4 3.0 Hz, J3,2 8.5 Hz, H-3), 3.50 (dd, 1H, H-40), 2.29
0
0
(m, 1H, OH2 ), 1.31 (d, 3H, J6 5 6.0 Hz, H3-60). 19: 8.01 (m, 4H, aromatic), 7.61–7.17 (m, 16H, aromatic), 5.79 (m, 1H,
0
0 0
OCH2CH_CH2), 5.64 (t, 1H, J2 ,1 +2 ,3 5.5 Hz, H-20), 5.56 (dd, 1H, J3 ,2 3.2 Hz, J3 ,4 8.5 Hz, H-30), 5.20 (d, 1H, J1 ,2
0 0
0
0
0
0
0
0
0
0
2.5 Hz, H-10), 5.18 and 5.07 (m, 2H, OCH2CH_CH2), 4.97 and 4.77 (ABq, 2H, JA,B 10.5 Hz, OCH2Ph), 4.77 and 4.63
(ABq, 2H, JA,B 11.3 Hz, OCH2Ph), 4.53 (m, 2H, H-6a and H-6b), 4.51 (d, 1H, J1,2 7.5 Hz, H-1), 4.18 and 4.09 (m, 2H,
OCH2CH_CH2), 4.06 (m, 1H, H-4), 4.02 (dq, 1H, J5 ,4 9.2 Hz, H-50), 3,80 (dd, 1H, J2,3 9.5 Hz, H-2), 3.75 (m, 1H, H-5),
0
0
3.52 (dd, 1H, J3,4 3.0 Hz, H-3), 3.48 (t, 1H, J4 ,3 +4 5 17.5 Hz, H-40), 1.95 (s, 3H, OCOCH3), 1.35 (d, 3H, J6 5 6.0 Hz,
H3-60), 1.30 (s, 9H, OC(CH3)3).
0
0
0
0
0 0