Synthesis of plant arabinogalactans

Abstract

Plant arabinogalactans consisting of a b-(1®6)-linked D-galactopyranosyl oligosaccharide backbone with a-(1®2)-L-arabinofuranosyl branches are synthesized based on the 1,2-anhydro galactopyranose technique, orthogonal (methoxydimethyl)methyl (MIP) and (2-naphthyl)methyl (NAP) protection strategy, and selective acylation or glycosylation method. The third method is the most simple and effective and it is also used for the synthesis of arabinogalactans composed of a b-(1®6)-linked D-galactopyranosyl oligosaccharide backbone with a-(1®3)-L-arabinofuranosyl branches.

Keywords

arabinogalactan / oligosaccharide synthesis

Introduction

One of the main problems in studying the biological functions of oligosaccharides is their limited availability in pure form with enough quantity from natural sources. Therefore, efficient chemical synthesis would make complex carbohydrates more accessible to general chemists and biochemists who want to keep pace with the exploding area of glycobiology [1]. Great effort has been devoted to the development of new strategies for glycosidic coupling, and methods for construction of glycosidic linkages have progressed considerably [2-5]. This review will only focus on the synthesis of plant arabinogalactans.

Arabinogalactans are often classified into three groups: arabino-4-galactans (Type I), arabino-3,6-galactans (Type II), and polysaccharides with arabinogalactan side chains (Type III) [6]. The latter types are also called the real pectins. Some arabinogalactans from certain sources show biological activity. One of the arabinogalactans which shows an activity on the complement system is an arabinogalactan from a hot water extract of the roots of the Chinese herb Angelica acutiloba [7]; such activity is not found in the arabinogalactan from larch wood [8]. An arabinogalactan isolated from the roots of Saposhnikova divaricata or Panex notoginseng has reticuloendothelial system activating properties [9]. The arabinogalactans with a b-(1®6)-linked galactopyranose backbone and a-(1®2)-linked arabinofuranose side chains may exist in Echinacea purpurea, and have immunomodulating activity [10]. Thus, studies on identification of the arabinogalactan structures with bioactivity are very important in glycoscience. Also, it is known [11] that an expanding field in phytobiology is the use of monoclonal antibodies for the study of complex carbohydrates present at plant cell surfaces. Antibodies generated against specific plant cell wall carbohydrates may serve as probes in identifying cognate structural elements in outer membrane saccharides of other plant species. The usefulness of this concept relies upon characterization of the epitopes recognized by the antibodies. Albersheim et al. reported [12] that a b-(1®6)-linked galactan containing at least three galactopyranosyl residues functionalized at 3-OH with an a-linked L-arabinofuranose unit is supposed to be the epitope recognized by the CCRC-M7 antibody. The production or the specificity elucidation of the monoclonal antibodies requires well-defined oligosaccharides which are the epitopes of the antigens. Besides, although the presence of 2,6- and 3,6-branched residues in arabinogalactans is well known, the exact structure of these saccharides remains to be established. Thus far, there has been no definite conclusion regarding the core structure or fragment of arabinogalactans with immunomodulating activity. Is the arabinogalactan with a b-(1®6)-linked galactopyranose backbone and a-(1®2)-linked arabinofuranose side chains active, or the arabinogalactan with the same backbone but with a-(1®3)-linked arabinofuranose branches active, or the arabinogalactan with the same backbone but with mixed a-(1®2)- and a-(1®3)-linked arabinofuranose branches active? To solve the above problems, the synthesis of a variety of model arabinogalactan structures, and consequent study of the biological activity of the synthetic samples are necessary.

Synthesis of arabinogalactans

There are some reports dealing with the synthesis of arabinogalactans which have been contributed mainly from three research groups led by van Boom [13], Lipták [14-17], and Kong [18-26]. They have developed quite different methods, i.e., the 1,2-anhydrogalactose-based method [13], the (methoxydimethyl)methyl (MIP) and (2-naphthyl)methyl (NAP) protection technique [14-17], and the selective acylation or glycosylation strategy [18-26].

Synthesis of arabinogalactans with 1,2-anhydrogalactose derivatives as the key intermediates

With 1,2-anhydrogalactose derivatives as the key intermediates, van Boom’s group have successfully accomplished the synthesis of three tetrasaccharides which are composed of the same b-(1®6)-linked D-galactopyranosyl trisaccharide backbone and one a-linked L-arabinofuranosyl branch attached at O-2, O-2', and O-2'' of the backbone, respectively. The synthetic routes for the tetramers with O-2 and O-2'-arabinofuranose branches are depicted in Scheme 1.

In the synthesized tetramers, the reducing end of the downstream galactose unit is functionalized with a dodecanoate spacer to enable conjugation to a solid support via a peptide linkage. In the synthesis, oxidative coupling of galactal is used for construction of the trisaccharide backbone. Thus, benzylation of 6-O-tert-butyldiphenylsilyl(TBDPS)-D-galactal 1 followed by 3,3-dimethyldioxirane (DMD)-mediated epoxidation gives the key intermediate, the fully protected 1,2-anhydrogalactose 2. Condensation of 2 with methoxycarbonylundecanol gives the acceptor b-galactoside 3 with a 2-free hydroxyl group, and the consequent NIS/TfOH catalyzed condensation with the donor thioethyl 2,3,4-tri-O-benzoyl-a-L-arabinofuranoside 4 affords the a-(1®2)-linked disaccharide 5. Desilylation of 5 (to 6) followed by ZnCl₂-catalyzed coupling with 2 yields the trisaccharide 7. Finally, desilylation of 7 and subsequent condensation with 2, followed by routine deprotection affords the target tetramer 8 with an a-linked arabinofuranose branch at O-2. Similarly, benzoylation of 3 (to 9) and consequent desilylation and condensation with 2 give the disaccharide acceptor 10, coupling of which, with 4, followed by desilylation, produces the trisaccharide acceptor 11. Condensation of 11 with 2 followed by deprotection affords the other tetramer 12 with an a-linked arabinofuranose branch at O-2'. Meanwhile, the third tetramer 13 with an a-linked arabinofuranose branch at O-2'' is also similarly synthesized.

A propitious feature of this approach is the concomitant formation of an unprotected 2-hydroxyl function during the couplings with 1,2-anhydrogalactose 2 as the donor, and hence direct arabinofuranosylation is possible. Theoretically, any arabinogalactan composed of a b-(1®6)-linked galactopyranose oligosaccharide backbone and a-(1®2)-linked arabinofuranose branches can be synthesized by this method. However, the use of an unnatural starting galactal derivative prepared from galactose through several steps limits its application in synthesizing higher arabinogalactans.

Synthesis of arabinogalactans based on the (methoxydimethyl)methyl (MIP) and (2-naphthyl)methyl (NAP) protection technique

Lipták’s group is the first to report [27] that the reaction of alkyl b-D-galactopyranosides with 2,2-dimethoxypropane in the presence of a catalytic amount of toluenesulfonic acid gives alkyl 3,4-O-isopropylidene- 6-O-(methoxydimethyl)methyl(MIP)-b-D-galactopyranosides at high yields. Since the products contain a free 2-hydroxyl group and a very easily removed 6-O-MIP group, they are successfully applied in the synthesis of 2-O-arabinofuranosylated b-(1®6)-galacopyranosyl oligosaccharides.

Synthesis of simple arabinogalactans based on (methoxydimethyl)methyl (MIP) technique

Some 2'-O-, 6'-O- and 2',6'-di-O-(a-L-arabinofuranosyl)-b-D-galactopyranosyl-(1®6)-galactoses are synthesized using the MIP technique by Lipták’s group, as shown in Scheme 2 [14]. In the synthesis, (1®6)-linked galactose disaccharide 14, prepared by coupling of acetobromogalactose with galactose 1,2:3,4-diacetonide, is used as the starting material. Thus, the disaccharide 14 is deacetylated and then treated with excess 2,2-dimethoxypropane in the presence of toluenesulfonic acid to give disaccharides 15 and 16 at a ratio of 4:1. An attempt to obtain a 2'-O-linked trisaccharide is not successful as arabinosylation of 15 with perbenzoylated a-L-arabinofuranosyl bromide using Hg(CN)₂ as the promoter produces a mixture of 2',6'-di-O-linked tetrasaccharide 17 (25%), 6'-O-linked trisaccharide 18 (25%), and 2'-O-linked trisaccharide 19 (5%), indicating that the MIP group is very labile even at weakly acidic coupling conditions. To avoid the cleavage of the MIP group, a rather basic condensation with AgOTf as the promoter in the presence of 2,4,6-trimethylpyridine is executed. It is found, however, that the condensation proceeds to a moderate yield giving two products, 20 and 21, at a ratio of 5:4. Compound 20 is the request trisaccharide and 21 is a trisaccharide dimer linked at 6'-O- through an isopropylidene acetal linkage, being able to transform to 19via mild acidic hydrolysis. Compounds 17, 18, and 19 are easily deprotected by routine methods to give the corresponding 2',6'-branched tetrasaccharide, 6'-branched trisaccharide, and 2'-branched trisaccharide, respectively. It seems that the MIP-containing acceptor 15 can be used in the synthesis of small arabinogalactans but its instability complicates the coupling reactions and decreases the coupling yields, and thus 15 is not a good glycosyl acceptor for the synthesis of higher arabinogalactans.

Synthesis of arabinogalactan hexasaccharide based on combination of MIP and benzyl protection

To overcome the instability of the MIP group during the coupling of 15 with the arabinose donor, Lipták et al. improved [15] the synthetic method to synthesize a hexasaccharide, a-L-Araf-(1®2)-b-D-Galp-(1®6)-b-D-Galp-(1®6)-[a-L-Araf-(1®2)-]b-D-Galp-(1®6)-D-Gal, the anticipated repeating unit of arabinogalactan from the cell culture of E. purpurea, as shown in Scheme 3.

In this synthesis the disaccharide 15 is not directly arabinosylated at O-2', instead, it is benzylated to block O-2' to obtain a fully protected disaccharide, and following with mild hydrolysis selectively removes the MIP group in the presence of isopropylidene groups to give the disaccharide acceptor 22 at a high yield. Meanwhile, hydrolysis of the fully protected disaccharide for removal of the acidic labile groups and then acetylation, selective 1-O-deacetylation and trichloroacetimidation afford the disaccharide donor 23. Condensation of the donor 23 with the acceptor 22 is carried out smoothly, giving the tetrasaccharide 24 with benzylated O-2' and O-2'''. Consequently, under neutral conditions hydrogenolysis to remove benzyl groups is executed smoothly, giving a tetrasaccharide acceptor with O-2' and O-2''' free hydroxyl groups. Subsequent coupling with the benzoylated arabinofuranosyl bromide donor in the presence of Hg(CN)₂ as the promoter affords the hexasaccharide 25 at a fair yield, and routine deprotection gives the target hexasaccharide.

This method prevents the difficulties caused by the MIP group due to its hydrolysis or participation in condensation reactions, and successfully makes use of the difference between MIP and the isopropylidene group for preparation of the disaccharide acceptor, thus it is an effective method for preparation of 2-branched arabinogalactans.

Synthesis of arabinogalactans based on combination of MIP and NAP protection

Similarly, Lipták et al. also successfully use (2-naphthyl)methyl (NAP) as the protective group of O-2' in combination [16] with the use of MIP at O-6'. The NAP group, like the benzyl group, can be removed by catalytic hydrogenolysis, but NAP is more sensitive and can be selectively removed in the presence of the latter [28]. Besides, the NAP group is very similar to 4-methoxybenzyl, but NAP is more resistant to acids, being stable under the acidic conditions used for removal of the isopropylidene groups. As shown in Scheme 4, the disaccharide 15 with a 2'-free hydroxyl group is readily (2-naphthyl)methylated with (2-naphthyl)methyl bromide and sodium hydride in DMF to give 26 at good yield. Then very mild hydrolysis of 26 cleaves the MIP group, giving the disaccharide acceptor 27 with a 6'-free hydroxyl group. Condensation of 27 with the acetylated galactosyl bromide in the presence of Hg(CN)₂ as the promoter affords the b-(1®6)-linked trisaccharide 28 in a moderate yield, and subsequent oxidative cleavage of the NAP group is achieved readily with DDQ in excellent yield, giving the trisaccharide acceptor 29 with a 2'-free hydroxyl group. Arabinosylation of 29 with 2,3,4-tri-O-acetyl-L-arabinofuranosyl trichloroacetimidate in the presence of TMSOTf as the catalyst and consequent deprotection give the target tetrasaccharide 30. Meanwhile, condensation of 29 with fully acetylated a-L-(1®5)-linked disaccharide trichloroacetimidate followed by deprotection affords the pentasaccharide 31 in high yields. Thus, the combination of MIP and the NAP protection method is effective for the synthesis of 2-branched arabinogalactans.

Synthesis of higher arabinogalactans based on a combination of MIP, NAP, and Benzyl protection

Lipták’s group further report [17] on the synthesis of higher arabinogalactans by the use of orthogonal MIP, NAP, and benzyl groups. In the synthesis, as shown in Scheme 5, the trisaccharide 28 is deacetonated, acetylated, 1-O-deacetylated, and trichloroacetimidated to give the trisaccharide donor 32 with NAP at O-2'. Meanwhile, the disaccharide acceptor 22 is galactosylated with acetobromogalactose in the presence of Hg(CN)₂ as the promoter to afford the trisaccharide 33. Zemplén deacetylation of 33 followed by reaction with 2,2-dimethoxypropane in the presence of catalytic toluenesulfonic acid produces the trisaccharide 34 with MIP at O-6'' and a free hydroxyl function at O-2''. Acetylation of 34 and consequent removal of the 6”-MIP afford the trisaccharide acceptor 35 with benzyl at O-2'. TMSOTf-catalyzed condensation of the acceptor 35 with the donor 32 yields the b-(1®6)-linked galactopyranose hexasaccharide framework 36 with NAP and benzyl at O-2'''' and O-2', respectively. Since the cleavage conditions for NAP and benzyl can be orthogonal, assembly of the arabinofuranose branch can be carried out separately, thus making either the same or different branches at O-2' and O-2''''. For example, oxidative removal of the NAP of 36 with DDQ produces the hexasaccharide acceptor 37 with a O-2'''' free hydroxyl group, and its condensation with 2,3.4-tri-O-acetyl-a-L-arabinofuranosyl trichloroacetimidate gives the heptasaccharide 38. Cleavage of the benzyl group of O-2' of 38 is smoothly carried out by catalytic hydrogenolysis, giving the heptasaccharide acceptor 39. Then, coupling of 39 with the fully acetylated a-L-(1®5)-linked disaccharide trichloroacetimidate affords the nonasaccharide 40, and consequent deprotection gives the target nonasaccharide 41. Similarly, the octasaccharide 42 with a-L-arabinofuranose branches at O-2' and O-2'''', and the nonasaccharide 43 with a-L-arabinofuranose and a-L-(1®5)-linked arabinofuranose disaccharide branches at O-2' and O-2'''', respectively, are successfully synthesized by the same method.

Thus, the combined use of orthogonal groups such as MIP, NAP, and benzyl is an effective technique for the synthesis of complex 2-branched arabinogalactans.

Synthesis of arabinogalactans based on selective deacetylation or galactosylation strategy

Kong’s group reports [29-31] that formation of sugar-sugar orthoesters consisting of a fully acylated mono- or disaccharide donor and a partially protected mono- or disaccharide acceptor is regioselective, and Lewis acid-catalyzed rearrangement of the orthoesters via RO-(orthoester)C bond cleavage gives a dioxolenium ion intermediate leading to 1,2-trans-glycosidic linkage. They also reveal [32] that the coupling reactions with acylated glycosyl trichloroacetimidates as the donors usually give orthoesters as the intermediates especially when the coupling is carried out at slowed rates, and this is successfully used in regio- and stereoselective syntheses of oligosaccharides. With a partially protected mono- or oligosaccharide as the acceptor, and 2-branched arabinogalactans are also readily prepared. Further, most of the glycosyl acceptors used in the synthesis are prepared by methanolysis to selectively remove the acetyl groups in the presence of benzoyl groups with methanolic hydrogen chloride obtained by mixing 0.2%-2% AcCl and MeOH-CH₂Cl₂ (a modification of a procedure proposed by Kochetkov et al.) [33].

Synthesis of 2- and 3-branched arabinogalactans based on selective glycosylation

The synthesis [18] of O-2 branched arabinogalactan tetrasaccharide by selective glycosylation is shown in Scheme 6. In the synthesis, the disaccharide 14 is hydrolyzed to remove isopropylidene groups, and consequent acetylation, selective 1-O-deacetylation, and trichloroacetimidate formation give the fully acetylated disaccharide trichloroacetimidate donor 44. Meanwhile, deacetylation of dodecyl tetra-O-acetyl-b-D-galactopyranoside 45 followed by isopropylidenation yields the monosaccharide acceptor 46 with 2,6-free hydroxyl groups. The coupling of the disaccharide donor 44 with the monosaccharide acceptor 46 is a regioselective reaction, giving the b(1®6)-linked trisaccharide 47 with a 2-free hydroxyl group at an acceptable yield. Then, direct condensation of 47 with benzoylated a-L-arabinofuranosyl trichloroacetimidate 48 [34, 35] produces the tetrasaccharide 49, and consequent deprotection affords the target O-2 branched tetrasaccharide glycoside 50.

Their study [19] also reveals that with 6-O-trityl-1,2-O-isopropylidene-a-D-galactopyranose 51, obtained readily from 1,2-O-isopropylidene-a-D-galactopyranose by tritylation, as the starting material, a 3-branched arabinogalactan tetrasaccharide is easily prepared, as shown in Scheme 7. Thus, selective coupling of the galactose acceptor 51 with the arabinose donor 48 gives the a-(1®3)-linked disaccharide 52. Removal of the trityl group with ferric trichloride hexahydrate [36] affords the disaccharide acceptor 53 with O-4 and O-6 free hydroxyl groups. Again, selective glycosylation of 53 with the disaccharide donor 44 is carried out smoothly, producing the tetrasaccharide 54, and subsequent deprotection gives the target O-3 branched tetramer.

Synthesis of higher 2-branched arabinogalactans by combination of selective glycosylation and deacetylation

Higher arabinogalactan oligosaccharides are also synthesized by a combination of selective glycosylation and deacetylation, as shown in Scheme 8. In the synthesis [20], several key building blocks, i.e. the glycosyl donors 56, 58, and 60, and the glycosyl acceptors 57 and 67, are prepared first. The three glycosyl donors have the same leaving group but with acetyl groups at O-6, O-2, and O-2,6, respectively, and the two glycosyl acceptors have free hydroxyl groups at O-6 and O-2,6 respectively.

The donor 56 is prepared from 3,4,6-tri-O-benzoyl-1,2-O-acetyl-D-galctopyranose 55 by O-1 selective deacetylation, and trichloroacetimidate formation, while 55 is obtained from galactose in a one-pot manner through 6-O-tritylation, benzoylation, and acetolysis. The donor 58 is readily produced from 1,2-O-isopropylidene-a-D-galactopyranose via benzoylation, hydrolysis, O-1,2 acetylation, O-1 selective deacetylation and trichloroacetimidate formation, and the donor 60 is obtained from 3,4-di-O-bezoyl-1,2-O-isopropylidene-6-O-trityl-a-D-galactopyranose 59 by acetolysis, O-1 selective deacetylation and trichloroacetimidate formation. The glycosyl acceptor 57 or 67 is easily obtained from coupling of the donor 56 or 60 with dodecyl alcohol followed by O-6 or O-2,6 selective deacetylation with 0.5% MeCOCl/MeOH. With these glycosyl donors and acceptors at hand, the 2-branched arabinogalactans are readily prepared. For example, coupling of the donor 56 with the acceptor 57 followed by O-6 selective deacetylation gives the disaccharide acceptor 61, and subsequent condensation of 61 with the donor 58 followed by O-2 selective deacetylation affords the trisaccharide acceptor 62 with an O-2'' free hydroxyl group. Similarly, coupling of the donor 60 with the acceptor 57 followed by O-2,6 deacetylation yields the disaccharide acceptor 63, while selective glycosylation of 63 with tetra-O-benzoyl-a-D-galactopyranosyl trichloroacetimidate 64 gives the (1®6)-linked trisaccharide acceptor, and consequent reaction with the arabinofuranose donor 48 affords the 2'-branched tetrasaccharide 65. Also, direct condensation of the trisaccharide acceptor 62 with 48 gives the 2''-branched 66. Meanwhile, selective glycosylation of the acceptor 67 with the donor 56 affords the (1®6)-linked disaccharide acceptor 68 with an O-2 free hydroxyl group. Condensation of the acceptor 68 with the arabinofuranose donor 48 gives the 2-branched trisaccharide 69, and the following O-6' deacetylation affords the trisaccharide acceptor 70. Then, coupling of 70 with the donor 60 followed by O-2'',6'' deacetylation yields the tetrasaccharide acceptor 71. Again, selective glycosylation of 71 with 64 produces the (1®6)-linked pentasaccharide acceptor 72 with an O-2'' free hydroxyl group. Direct condensation of 72 with the arabinofuranose donor 48 followed by deprotection affords the 2,2''-branched arabinogalactan hexasaccharide glycoside 73. It is seen from the description above that selective glycosylation in combination with selective deacetylation is certainly an efficient method for the synthesis of 2-branched arbinogalactan oligosaccharides.

Synthesis of 2-branched arabinogalactans by sequential selective O-6 and O-2 deacetylation

Further study [21, 22] reveals that not only can the benzoate and acetate groups be differentiated under the acidic condition of MeCOCl/CH₃OH/CH₂Cl₂ (1∶50∶50), but the O-6 acetyl and the O-2 acetyl groups can also be differentiated under the much milder condition of MeCOCl/CH₃OH/CH₂Cl₂ (1∶500∶500), where the former is removed under this condition without affecting the latter and the benzoate groups. This finding greatly simplifies the synthesis of 2-branched arabinogalactans since the use of orthogonal protective groups is avoided.

As shown in Scheme 9, TMSOTf-promoted condensation of 60 with 4-methoxyphenol followed by selective O-6 selective deacetylation gives the glycosyl acceptor 74. Then coupling of 74 with 64 gives the b-(1®6)-linked disaccharide glycoside, and the following oxidative cleavage of the O-1 MP group and trichloroacetimidate formation afford the disaccharide donor 75. Reaction of 75 with 4-methoxyphenol 3,4,6-tri-O-benzoyl-b-D-galactopyranoside 76, prepared from 56 through its condensation with 4-methoxyphenol followed by selective O-6 selective deacetylation, affords a trisaccharide, and consequent O-2 selective deacetylation with MeCOCl/MeOH/CH₂Cl₂ (1∶50∶50) gives the trisaccharide acceptor 77 with an O-2' free hydroxyl group. Condensation of 77 with the a-(1®5)-linked arabinofuranose disaccharide trichloroacetimidate 78 [34, 35] followed by routine protection yields the free pentasaccharide glycoside 79. Meanwhile, coupling of the glycosyl acceptor 74 with the donor 56 gives the disaccharide 80 and the following O-6' selective deacetylation affords the disaccharide acceptor 81 with an O-6' free hydroxyl group. Then reaction of 81 with the disaccharide donor 75 yields the b-(1®6)-linked tetrasaccharide 82, and consequent selective O-2,2'' deacetylation, condensation with the arabinofuranose disaccharide donor 78, and deprotection produce the target 2,2''-branched arabinogalactan octasaccharide 83. More complex arabinogalactans can also be prepared by this technique. For example, coupling of the trisaccharide acceptor 77 with the arabinofuranose donor 48 gives the tetrasaccharide 84, and the following oxidative cleavage of O-1 MP and trichloroacetimidate formation afford the tetrasaccharide donor 85. Meanwhile, coupling of the disaccharide acceptor 86, prepared from coupling 60 with 76 followed by O-6' selective deacetylation, with the tetrasaccharide donor 85 and consequent O-2' selective deacetylation produces the hexasaccharide acceptor 87. Finally, condensation of 87 with the arabinose disaccharide donor 78 followed by deprotection produces the octasaccharide glycoside 88 with structurally different branches at O-2' and O-2'''.

It is seen from the description above that the technique based on sequential O-6 and O-2 selective deacetylation gives the same outcome that selective glycosylation makes, however, the former method is more readily handled in large quantities, for general chemists.

Synthesis of higher 3-branched arabinogalactans by 3,6-selective protection of galactoside in combination with selective glycosylation

3-Arabinofufanose branched arabinogalactans are synthesized [23] based on a 3,6-selective protection technique [37, 38] in combination with selective glycosylation, as shown in Scheme 10. In the synthesis, detritylation of isopropyl 2,4-di-O-benzoyl-3-O-tert-butyldimethylsilyl-6-O-trityl-1-thio-b-D-galactopyranoside 89 [37, 38] with FeCl₃ hexahydrate [36], followed by condensation with tetra-O-benzoyl-a-D-galactopyranosyl trichloroacetimidate 64 gives the b-(1®6)-linked disaccharide 90. Desilylation of 90 and consequent coupling with the arabinose donor 48 afford the trisaccharide donor 91. Meanwhile, the glycoside acceptor 93 with 3,4-free hydroxyl groups is prepared via selective O-2 benzoylation of methyl 4,6-O-isopropylidene-a-D-galactopyranoside (to 92) and the following deisopropylidenation and selective 6-O-tert-butyldiphenylsilylation. Then, selective coupling of 93 with the arabinose donor 48 affords the (1®3)-linked disaccharide 94, and the following desilylation and selective coupling with phenyl 2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenyl-1-thio-b-D-galactopyranoside 95 give the b-(1®6)-linked trisaccharide 96. Acetylation of 96 followed by desilylation affords the trisaccharide acceptor 97. Finally, coupling of 97 with the trisaccharide donor 91 followed by deprotection yields the target 3- and 3''-branched arabinogalactan hexasaccharide 98. It is seen from the above descriptions that this method successfully uses selective glycosylation, however, it needs some special silyl reagents and a rather complex procedure.

Synthesis of higher 3-branched arabinogalactans by O-3 selective allylation of galactoside in combination with selective O-6 deacetylation

A more accessible method [24, 25] based on 3-selective allylation of galactoside in combination with O-6 deacetylation is developed, as shown in Scheme 11. The key building block 4-methoxybenzyl 3-O-allyl-2,4-di-O-benzoyl-b-D-galactopyranoside 99 is obtained in high yield from 4-methoxybenzyl tetra-O-acetyl-b-D-galactopyranoside via Zémplen deacetylation, dibutylstannylene-mediated 3-O-selective allylation [39], 6-O-tritylation, benzoylation, and detritylation. Coupling of 99 with the fully benzoylated galactopyranosyl trichloroacetimidate 64 and consequent oxidative cleavage of O-1 MP and trichloroacetimidate formation produce the disaccharide donor 100. Then, condensation of the glycosyl acceptor 76 with 100 gives the trisaccharide 101 and the following deallylation affords the trisaccharide acceptor 102. Direct coupling of 102 with the arabinose donor 48 and consequent transformation of the 1-O-MP to 1-O-trichloroacetimidate produce the tetrasaccharide donor 103. Meanwhile, coupling of 99 with 56 followed by activation of the anomeric center yields the disaccharide donor 104, and consequent condensation with 76 followed by deallylation gives the trisaccharide 105. Reaction of 105 with the arabinobiose trichloroacetimidate 78 followed by selective O-6'' deacetylation yields the pentasaccharide acceptor 106. Finally, condensation of 106 with 103 followed by deprotection yields the 3'-arabinobiose- and 3''''-arabinose-branched target nonasaccharide 107. By the same strategy, 3'-arabinose- and 3''''-arabinobiose-branched nonasaccharide 108, 3'-, 3''''-arabinose-branched octamer 109, 3'-, 3''''-, 3⁸'-branched dodecamer 110, and 3'-, 3''''-, 3⁸'-, 3¹²'-branched sixteenmer 111 are also concisely synthesized. It can be seen from the descriptions above that this method is very effective and concise for the synthesis of 3-branched arabinogalactans in large quantities.

Synthesis of arabinogalactans with both 2- and 3-arabinose branches

As shown in Scheme 12, concise synthesis of the arabinogalactans with both a-(1®3)- and a-(1®2)-linked arabinose branches is achieved [26]. Isopropyl 1-thio-b-D-galactopyranoside, prepared readily by coupling of penta-O-acetyl-b-D-galactopyranose with isopropyl thiol and subsequent deacetylation, is chosen as the starting material. Its selective 3-O-allylation via dibutyltin complex and the following 6-O-tritylation, 2,4-di-O-benzoylation and 6-O-detritylation give the glycosyl acceptor 112 at a satisfactory yield. Condensation of 112 with perbenzoylated galactopyranosyl trichloroacetimidate 64 affords the disaccharide donor 113. Then, condensation of 113 with the disaccharide acceptor 81 followed by selective 2-O-deacetylation gives the tetrasaccharide acceptor 114. Condensation of 114 with the arabinose donor 48 produces the pentasaccharide 115, and subsequent deallylation affords the acceptor 116. Coupling of 116 with the arabinose donor 48 followed by deacylation give the target hexasaccharide 117 consisting of a b-(1®6)-linked galactopyranosyl tetrasaccharide backbone and a-L-arabinofuranose side chains at O-2 and O-3'', respectively.

The octamer 121, dodecamer 122, and twentymer 123 are also effectively synthesized with three tetrasaccharide building blocks, i.e., the 3-branched acceptor 118, the 2-branched donor 119, and the 3-branched donor 120; they are prepared readily by the developed method. In the synthesis, the oligosaccharide chain is elongated from the non-reducing end. Thus, coupling of 118 with 119 affords the octamer 121, and consequent deprotection gives the free 4-methoxyphenyl glycoside of octasaccharide. Selective 6'''''-O-deacetylation of 121 is carried out under stronger acidic conditions, i.e., 1∶50∶50 MeCOCl/MeOH/CH₂Cl₂, because of the weaker reactivity of the long chain, and the following coupling with the 3-branched donor 120 yields 122, deprotection of which gives the free glycoside of dodecasaccharide. Starting from 122, reiteration of selective deacetylation, coupling (with 119), and deprotection produces the target twentymer glycoside 123.

It can be seen from the above statements that this method is simple, highly regio- and chemoselective, and can be used in the preparation of structurally different arabinogalactans required in glycobiology research. With the synthesized arabinogalactans at hand, the study of structure-bioactivity is readily performed. Preliminary mice testing results (unpublished) indicate that some synthesized arabinogalactans show strong immunomodulating activity. It is expected that a conclusion on structure-bioactivity of arabinogalactans will be drawn in the near future.

References

[1] Bertozzi C R, Kiessling L L. Chemical glycobiology. Science, 2001, 291: 2357-2364

[2] Kong F. Regio- and stereoselective synthesis of oligosaccharides with unprotected or lightly protected sugars as glycosyl acceptors. Curr Org Chem, 2003, 7(9): 841-865

[3] Davis B G. Recent development in oligosaccharide synthesis, J Chem Soc Perkin Trans 1, 2000, (14): 2137-2160

[4] Schmidt R R, Kinzy W. Adv Carbohydr Chem Biochem, 1994, 50: 21-125

[5] Toshima K, Tatsuta K. Recent progress in O-glycosylation methods and its application to natural products synthesis. Chem Rev, 1993, 93: 1503-1531

[6] Clarke A E, Anderson R L, Stone B A. Form and function of arabinogalactans and arabinogalactan-protein. Phytochemistry, 1979, 18: 521-528

[7] Yamada H, Kiyohara H, Cyong J C, Otsuka Y. Studies on polysaccharides from Angelica acutiloba-IV, characterization of anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa. Mol Immunol, 1985, 22: 295-304

[8] Kiyohara H, Yamada H, Cyong J C, Otsuka Y. Characterization and function of arabinogalactans. J Pharmacobio-Dyn, 1986, 9: 339-346

[9] Shimizu N, Tomoda M, Gonda R. Studies on the constituents of edible and medicinal plants. Chem Pharm Bull (Tokyo), 1989, 37: 1329-1332

[10] Egert D, Beuscher N. Studies on antigen specificity of immunoreactive arabinogalactan proteins extracted from Baptisa tinctoria and Echinacea purpurea. Planta Med, 1992, 58: 163-165

[11] Hoson T. Structure and function of plant cell walls: immunological approaches. Int Rev Cytol, 1991, 130: 233-268

[12] Steffan W, Kovac P, Albersheim P, Darvill A G, Hahn M G. Characterization of a monoclonal antibody that recognizes an arabinosylated β-1-6-galactan epitope in plant complex carbohydrates. Carbohydr Res, 1995, 275: 295-307

[13] Timmers C M, Wigchert S C M, Leeuwenburgh M A, van der Marel G A, van Boom J H. Synthesis of tetrameric arabinogalactans based on 1,2-anhydrosugars. Eur J Org Chem, 1998, 1: 91-97

[14] Borbás A, Jánossy L, Lipták A. Scope and limitation of the application of the (methoxydimethyl)methyl group in the synthesis of 2'-O-, 6'-O- and 2',6'-di-O-(α-L-arabinofuranosyl)-β-D-galactopyranosyl-(1→6)-D-galactoses. Carbohydr Res, 1999, 318: 98-109

[15] Csávás M, Borbás A, Jánossy L, Batta G, Lipták A. Synthesis of the α-L-Araf-(1→2)-β-D-Galp-(1→6)-β-D-Galp-(1→6)-[α-L-Araf-(1→2)]-β-D-Galp-(1→6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan. Carbohydr Res, 2001, 336: 107-115

[16] Csávás M, Borbás A, Szilagyi L, Lipták A. Successful combination of (methoxydimethyl)methyl (MIP) and (2-naphthyl)methyl (NAP) ethers for the synthesis of arabinogalactan-type oligosaccharides. Synlett, 2002, 6: 887-890

[17] Csávás M, Borbás A, Jánossy L, Lipták A. Synthesis of an arabinogalactan-type octa- abd two isomeric nonasaccharides. Suitable tuning of protecting groups. Tetrahedron Lett, 2003, 44: 631-635

[18] Du Y, Pan Q, Kong F. Synthesis of a tetrasaccharide representing minimal epitope of arabinogalactan. Carbohydr Res, 2000, 323: 28-35

[19] Du Y, Pan Q, Kong F. Synthesis of 3-O-arabinosylated (1-6)-galactan. J Carbohydr Chem, 2001, 20: 297-306

[20] Ning J, Yi Y, Yao Z. An efficient method for the synthesis of 2,6-branched galacto-oligosaccharides and its application to the synthesis of three tetrasaccharides and a hexasaccharide related to the arabinogalactans (AGs). Synlett 2003, 14: 2208-2212

[21] Zeng Y, Li A, Kong F. A concise synthesis of arabinogalactan with β-(1→6) galactopyranose backbone and α-(1→2) arabinofuranose side chains. Tetrahedron Lett, 2003, 44: 8325-8329

[22] Li A, Zeng Y, Kong, F. An effective synthesis of arabinogalactan with β-(1→6) galactopyranose backbone and α-(1→2) arabinofuranose side chains. Carbohydr Res, 2004, 339: 673-681

[23] Gu G, Du Y, Kong, F. Synthesis of a hexasaccharide that relates to the arabinogalactan epitope. Carbohydr Res, 2001, 336: 99-106

[24] Li A, Kong F. Concise syntheses of arabinogalactans consisting of β-(1→6)-linked galactopyranosyl backbone and α-(1→3)-linked arabinofuranosyl side chains. Chin J Chem, 2005, 23: 7-10

[25] Li A, Kong, F. Syntheses of arabinogalactans consisting of β-(1→6)-linked D-galactopyranosyl backbone and α-(1→3)-linked L-arabinofuranosyl side chains. Carbohydr Res, 2004, 339: 1847-1856

[26] Li A, Kong, F. Concise syntheses of arabinogalactans with β-(1→6)-linked galactopyranose backbones and α-(1→3)- and α-(1→2)-linked arabinofuranose side chains. Biorg Med Chem, 2005, 13: 839-853

[27] Lipták A, Fugedi P, Kerekgyarto J, Nanasi P. Preparation of mixed-acetal derivatives of carbohydrates by acetal-exchange reaction. Carbohydr Res, 1983, 113: 225-231

[28] Gaunt M J, Yu J, Spencer J B. Rational design of benzyl-type protecting groups allows sequential deprotection of hydroxyl groups by catalytic hydrogenolysis. J Org Chem, 1998, 63: 4172-4173

[29] Wang W, Kong F. New method for regio- and stereoselective synthesis of oligosaccharides. J Org Chem, 1998, 63: 5744-5745

[30] Wang W, Kong F. A highly convergent synthesis of the elicitor hexasaccharide. Tetrahedron Lett, 1998, 39: 1937-1940

[31] Wang W, Kong F. Regio- and steroselective synthesis of mannose-containing oligosaccharides. Angew Chem Int Ed, 1999, 38: 1247-1250

[32] Kong F. Recent studies on reaction pathways and applications of sugar orthoresters in synthesis of oligosaccharides. Carbohydr Res, 2007, 342: 345-373

[33] Byramova N E, Ovchinnikov M V, Backinowsky L V, Kochetkov N K. Selective removal of O-acetyl groups in the presence of O-benzoyl groups by acid-catalyzed methanolysis. Carbohydr Res, 1983, 124: c8-c11

[34] Du Y, Pan Q, Kong F. Regio- and steroselective synthesis of 1-5-linked arabinofuranosyl oligosaccharides using unprotected furanoside donors. Synlett, 1999:, 1648-1652

[35] Du Y, Pan Q, Kong, F. A facile synthesis of 1-5-linked arabinofuranosyl oligosaccharides. Carbohydr Res, 2000, 329: 17-24

[36] Ding X, Wang W, Kong F. Selective O-detritylation of sugar derivatives. Carbohydr Res, 1997, 303: 445-448

[37] Du Y, Zhang M, Kong F. Concise synthesis of 3,6-branched oligosaccharides. Org Lett, 2000, 2: 3797-3800

[38] Du Y, Zhang M, Kong F. Facile synthesis biologically important pentasaccharides. Tetrahedron, 2001, 57: 1757-1763

[39] Kong F, Lu D, Zhou S. Synthesis and conformation of 1,3-anhydro-2,4,6-tri-O-benzyl-D-galactose. Carbohydr Res, 1990, 198: 141-147

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg