Synthetic Study of Bio-functional Glycans

Abstract

The molecular structures responsible for the immune functions of complex glycans were unraveled by synthetic studies. We focused on developing efficient methods for synthesizing glycans and conducting diverse chemical syntheses of these compounds, to identify the molecular structures responsible for activating or modulating innate immunity. Many natural glycans contain multiple active structures, potentially leading to emergent higher-order functions through their synergistic interactions. Therefore, by employing a conjugation-based approach, we successfully created immune-regulating complex glycoconjugates.

21.1 Introduction

Glycans play pivotal roles in various biological events, such as intercellular interactions, protein quality control, and activation or modulation of immune responses. They are also closely associated with the onset of many diseases. Natural glycans exhibit structural diversity and heterogeneity, often harboring several recognition sites for various enzymes and lectins. By synthesizing homogeneous oligosaccharides chemically and subjecting them to bioactivity assays, we have contributed to identifying glycan structures responsible for recognition (active units).

In this paper, we describe our synthetic studies on bacterial-derived glycoconjugates that activate or modulate innate immunity, focusing on the immunomodulatory functions in parasite and symbiont-derived lipid A.

We also describe a diacetyl strategy developed for synthesizing NHAc-containing glycans and its application in synthesis of asparagine-linked glycoprotein glycans (N-glycans).

Many natural glycans have multiple units responsible for recognition, facilitating the manifestation of higher-order functions through various unit actions, including multivalent interactions. Consequently, combining these active units enables the creation of higher-order functional molecules. Successful generation of new immunomodulatory compounds was achieved by conjugating synthetic glycans and glycan dendrimers.

21.2 Development of Innate Immune Regulatory Molecules Based on Host-Bacteria Interactions and Their Application as Novel Adjuvants

We have been investigating the synthesis of immunostimulatory glycoconjugates derived from bacteria, such as bacterial cell wall peptidoglycans and lipopolysaccharides (LPS) from gram negative bacteria, to elucidate the mechanism of action in innate immunity [1, 2].

In canonical Escherichia coli LPS, the lipid A portion 1 (Fig. 21.1) binds to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, activating multiple downstream pathways, including two primary pathways alongside the caspase pathway, thereby activating the acquired immune system [3]. However, this activation also leads to toxic effects, such as lethal inflammation, due to its potent inflammatory activity. Extensive structure–activity relationship studies, including those conducted by our group, led to the discovery that mono-phosphoryl lipid As (MPL) including 2 and 3 exhibit mild immune-potentiating effects with low toxicity [4,5,6]. The 3D-MPL 3 developed by GlaxoSmithKline has proven effective as an adjuvant (a substance that enhances the efficacy of vaccines) for viral vaccines [6] and has been utilized in several vaccines in practical applications [7, 8]. Meanwhile, the development of mucosal vaccines capable of efficiently inducing immunity at the mucosal entry points of pathogens has been underway. However, MPL does not activate mucosal immunity, leaving the exploration of safe adjuvants for mucosal vaccines unexplored.

Fig. 21.1

Structures of lipid A

Therefore, we focused on bacteria that inhabit or parasitize mucosal tissues such as the oral cavity, stomach, and intestines. We hypothesized that these bacteria express molecules possessing immunomodulatory effects due to co-evolution with the host. By synthesizing lipid A and LPS partial structures from symbiotic and parasitic bacteria, we demonstrated that these structures exhibit characteristic immune-enhancing or immunomodulatory effects with low toxicity.

Parasitic bacteria such as Helicobacter pylori, associated with gastric ulcers, and the periodontal pathogen Porphyromonas gingivalis possess characteristic lipid A structures, which differ from the canonical E. coli lipid A 1 (Fig. 21.1). They contain longer chain fatty acids but in smaller numbers compared to E. coli lipid A, and mono-phosphoryl lipid A structures, some of which are ethanolamine modified. We developed a diversity-oriented synthetic strategy (Fig. 21.2), in which fatty acids are introduced sequentially to the common synthetic precursor, to synthesize ten structural variations of lipid A and Kdo-lipid A 4–10 [9, 10].

Fig. 21.2

Synthetic scheme of lipid A and Kdo-lipid A from parasitic bacteria

For the α-selective glycosylation reaction of Kdo, we devised Kdo donor 13 wherein the 6-membered ring was constrained into a boat-like conformation to promote the glycosyl acceptor’s attack from the α-orientation (Fig. 21.2) [9, 11]. However, the β-elimination reaction considerably occurred, leading to formation of glycal 17 due to the distortion of the 6-membered ring in the boat-like conformation. Efficient mixing using a microflow reactor promoted intermolecular glycosylation reactions and suppressed glycal formation to afford trisaccharide 16 in good yields (Fig. 21.3).

Fig. 21.3

Microflow glycosylation with Kdo donor

These compounds did not exhibited potent inflammatory effects; Kdo-lipid A 6a and 6b were found to act as an antagonist of TLR4-MD2, whereas ethanol amine modified lipid A 4b and 5b showed weak agonistic activity. These results revealed that Kdo-lipid A 6a and 6b plays an essential role in H. pylori LPS, contrary to the conventional understanding that lipid A is the active component of LPS. These results suggest that LPS derived from parasitic bacteria contributes to evading the bactericidal effects caused by acute inflammation. Conversely, all compounds induced the production of IL-18, associated with chronic inflammation. This highlights the importance of parasitic bacterial LPS as a molecule regulating host immune responses and suggests its involvement in chronic inflammation while circumventing acute inflammation.

Alcaligenes faecalis, known as an opportunistic Gram negative bacterium, was found to inhabit the gut-associated lymphoid tissue (GALT) known as Peyer’s patches, playing a crucial role in maintaining homeostasis. In collaboration with Kiyono and Kunisawa, we extracted LPS fractions from dried A. faecalis and found that A. faecalis LPS fraction exhibited no harmful effects but significantly promoted the production of IgA antibodies comparable to the toxic E. coli LPS [12]. Given that these effects were TLR4-dependent, A. faecalis lipid A was anticipated as a promising and safe adjuvant candidate. We then determined the structure of the A. faecalis LPS in collaboration with Molinaro and Di Lorenzo. We also found that the lipid A from A. faecalis is a mixture comprising compounds 18 ~ 20 with 4–6 fatty acid chains [13] (Fig. 21.4).

Fig. 21.4

Structures of Alcaligenes faecalis lipid A

We designed the key disaccharide intermediate 14 with orthogonal protecting group patterns applicable to various lipid A syntheses with different acyl patterns and established a diversity-oriented strategy for lipid A synthesis (Fig. 21.5). Each protecting group of disaccharide intermediate 14, 1-O-allyl, 2-N-allyloxycarbonyl (Alloc), 2′-N-2,2,2-trichloroethoxycarbonyl (Troc), 3′-O-p-methoxybenzyl (MPM), and 4′,6′-benzylidene, could be selectively removed to sequentially introduce acyl and phosphate groups at appropriate positions. Figure 21.5 illustrates a detailed synthetic scheme of A. faecalis lipid A 20 starting from intermediate 14. Fatty acid 21 was introduced at the 3-position of 14 in the presence of MNBA to obtain 22. Subsequently, removal of the 2′-N-Troc group of 22 using Zn-Cu couple, followed by acylation of the free amino group with fatty acid 23 using MNBA, was performed. Next, removal of the 2-N-Alloc group of 24 using Pd(PPh₃)₄ and TMSDMA, followed by the introduction of fatty acid 25 to the free 2-amino group using HATU, yielded 26. After cleaving the 3-position MPM group via oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), fatty acid 27 was introduced using MNBA to obtain 28. Subsequently, removal of the 4′,6′-O-benzylidene group of 28 using trifluoroacetic acid (TFA) followed by the selective introduction of a trityl (Tr) group at the 6′-position was conducted. Then, removal of the 1-O-allyl group of 29 led to the formation of 1,4′-dihydroxy 30. Simultaneous phosphitylation of the 1- and 4′-positions using phosphoramidite followed by DMDO oxidation yielded the desired 1,4′-O-diphosphate 31. Finally, all benzyl-type protecting groups were removed by catalytic hydrogenolysis to give A. faecalis lipid A 20 [13].

Fig. 21.5

Synthesis of hexa-acylated Alcaligenes faecalis lipid A

Among the synthesized compounds, only the hexa-acylated A. faecalis lipid A 20 exhibited immune-activating activity. Confirming its similarity to the extracted A. faecalis LPS, it was verified that A. faecalis lipid A 20 opposed the activity of A. faecalis LPS. Further in vivo experiments using mice demonstrated that A. faecalis lipid A 20 exhibited useful adjuvant effects without toxicity, enhancing antigen-specific IgA and IgG production and reinforcing defensive immunity via Th17 [13,14,15,16,17]. Particularly, enhanced antigen-specific IgA and IgG production was observed in mice administered with antigen and A. faecalis lipid A 20 adjuvant via intranasal administration. The effectiveness of 20 as a safe intranasal vaccine adjuvant was demonstrated in a pneumococcal infection model [15]. Lipid A 20 is anticipated to be a safe and promising adjuvant capable of activating mucosal and systemic immunity.

21.3 Synthetic Studies of Sialylated N-glycans by Diacetyl Strategy

Asparagine-linked (N-linked) glycans in glycoproteins (N-glycans) are oligosaccharides present in both eukaryotes and some prokaryotes with a wide range of structural variations. These glycans fall into three primary categories: high-mannose type, complex type, and hybrid type. Even within specific glycosylation sites, N-glycans typically display considerable heterogeneity. Complex N-glycans hold pivotal roles in diverse biological mechanisms and diseases, influencing glycoprotein dynamics, cell development, immune responses, and the progression of cancer invasion.

We developed a diacetyl strategy by temporarily converting NHAc to diacetyl imide (NAc₂) for the synthesis of acetamide (NHAc) containing glycans [18], since protected glycans containing NHAc tend to form intermolecular hydrogen bonds in organic solvents, greatly reducing the reactivity of glycosylation. The diacetyl strategy presents two advantages for oligosaccharide synthesis. The NAc₂ protection of NHAc substantially enhances glycosylation reactions, resulting in increased yields. Moreover, NAc₂ can be readily converted to NHAc by removing one acetyl group under mild basic conditions.

The disialylated tetrasaccharide (Neu5Ac(α2,3)Gal(β1,3)[Neu5Ac(α2,6)]GlcNAc), a structural motif present in the N-glycans of human Factor X and fetuin, was successfully synthesized using the diacetyl strategy [19]. The impact of NAc₂ was immense (Fig. 21.6). Glycosylation reactions between two sialyl disaccharides 32 and 33 with NHAc at the C5 position of sialic acid residues did not progress at all. However, the reactivity of NAc₂-protected sialyl fragments 35 and 36 significantly improved, resulting in the quantitative formation of the desired tetrasaccharide 37.

Fig. 21.6

Synthesis of disialylated tetrasaccharide

We then describe the synthesis of a core fucose-containing disialylated N-glycan, and two asymmetrically deuterated sialyl N-glycans, wherein one of the terminal sialic acids has been deuterium-labeled by replacing its NHAc.

We utilized the diacetyl strategy in synthesizing the non-reducing-end tetrasaccharide within the core-fucosylated N-glycan [20] (Fig. 21.7). Glycosylation of the NAc₂-protected sialylated disaccharide donor 39 with the disaccharide acceptor 40 proceeded rapidly at 0 °C, yielding the desired tetrasaccharide 42 at 96% yield. In contrast, glycosylation between the NHAc-containing sialyl disaccharide donor 38 and the disaccharide acceptor 40 only afforded the desired tetrasaccharide 41 at 52% yield, even after increasing the temperature to room temperature.

Fig. 21.7

Synthesis of core-fucosylated N-glycan

Another pivotal aspect in synthesizing the core fucose-containing glycan was the solvent selection for the glycosylation process between the reducing-end tetrasaccharide 42 and the non-reducing-end tetrasaccharide 43. Using ether-based solvents, particularly cyclopentyl methyl ether (CPME), yielded the targeted octasaccharide 44 at a 91% yield. The employment of ether solvents likely prolonged the stability of the intermediate oxocarbenium ion through coordination, albeit with a moderate stereoselectivity in glycosylation (α/β = 3/1). After removing the benzylidene group from the obtained octasaccharide, the α-isomer 45 was separated. Subsequent glycosylation at the 6th position of the branched mannose in 45 displayed a high yield of the desired product 46 when CPME was used as the solvent. However, α/β selectivity remained poor, resulting in a 1/1 mixture for compound 46. The global deprotection of 46 was then investigated. The allyl ester in 46 was cleaved using a Pd catalyst. The resulting carboxylic acid was then treated with aqueous LiOH to remove Troc, acyl groups, and methyl esters, and subsequent N-acetylation and separation of the α and β anomers by HPLC afforded 47. All benzyl-type protecting groups were removed by catalytic hydrogenolysis, resulting in the core fucose-containing N-glycan 48.

Next, we applied the diacetyl strategy to the synthesis of two asymmetrically deuterated sialyl N-glycans, 58 and 59 (Fig. 21.8) [21]. Using the deuterium-labeled N-glycan 58, we demonstrated the preferential cleavage of sialic acid on the α1,3 branch over the α1,6 branch by neuraminidase derived from the H1N1 influenza virus [22].

Fig. 21.8

Synthesis of asymmetrically deuterium-labeled biantennary N-glycans

In the synthesis of deuterated N-glycans 58 and 59, glycosylation was initially performed at the 3-position of the branching mannose in trisaccharide 51 using sialyl tetrasaccharides 49 or 50. Subsequently, glycosylation occurred at the 6-position of the branching mannose. We applied the remote participation method, previously described by Kim et al., for α-mannosylation. This technique involves acyl protection of the mannosyl donors at the O-3 and O-6 positions to enhance α-selectivity (Fig. 21.9).

Fig. 21.9

Remote participations from 3 and 6 positions of mannose

Ether solvent effect was also used in the glycosylation between 49 and 51. Using a stoichiometric amount of TMSOTf in Et₂O, the desired heptasaccharide 52 was obtained in 71% yield with perfect α-selectivity. After removal of benzylidene in 52, the glycosylation of the resulting 54 with the azide sialyl tetrasaccharide 50 was then investigated. Due to the poor solubility of 54 in Et₂O, the [7 + 4] glycosylation between 54 and 50 was conducted in a mixed solvent system of Et₂O/CH₂Cl₂ = 1/1. The desired undecasaccharide 56 was thus obtained in 85% yield with perfect α-selectivity.

Glycosylation of the azide-containing sialyl tetrasaccharide 50 with 51 was also carried out in Et₂O. The subsequent deprotection of benzylidene in 53 afforded 55 in good yield. Glycosylation between 55 and 49 under similar conditions afforded 57 in 54% yield (BRSM: 63%).

The desired deuterated N-glycans, 58 and 59, were synthesized from 56 and 57 through the incorporation of a deuterated acetyl group, followed by a global deprotection process. During the alkaline treatment to eliminate acyl and Troc groups, a deuterium-hydrogen exchange occurred to cause a reduction in the deuterium ratio of 58–42% and that of 59–63%, respectively.

Within naturally occurring N-glycans, the tetrasialylated N-glycan holds significance in assessing the effects of multivalency. The fully sialylated tetraantennary N-glycan 64 was synthesized by a similar approach to that of 58 and 59 (Fig. 21.10) [21]. The glycosylation between trisaccharide 51 and the heptasaccharide donor 60, in a mixed solvent of Et₂O/CH₂Cl₂ = 1/1, gave the desired decasaccharide with complete α-selectivity. Subsequent cleavage of the benzylidene group led to a 33% yield of 61 (BRSM: 49%) in two steps. The choice of Lewis acid, solvent, and temperature played a pivotal role in the subsequent glycosidation between decasaccharide 61 and heptasaccharide donor 62. Glycosylation of 61 and 62 was accomplished using TBDPSOTf at 0 °C in a high-ether ratio mixed solvent (Et₂O/CH₂Cl₂ = 5/1), resulting in a 36% yield of compound 63. Upon the deprotection of 63 and Fmoc introduction, the fully sialylated tetraantennary N-glycan 64 was obtained.

Fig. 21.10

Synthesis of tetraantennary sialyl N-glycan

As described above, the utilization of the diacetyl strategy led to the successful synthesis of various sialyl N-glycans, marking the world’s first chemical synthesis of a tetraantennary sialyl N-glycan.

21.4 Synthesis of Glycan Dendrimers and Their Applications to Biofunctional Studies

The interaction between glycans and glycan-binding proteins is typically weak, except for certain innate immune receptors. Polysaccharides and numerous glycans found on cell surfaces possess multiple binding sites, playing a role in multivalent interactions between glycans and glycan-binding molecules like lectins. This represents a pivotal aspect of glycan function, where high avidity and significant selectivity are achieved through multivalent interactions. Consequently, there have been a growing interest in developing multivalent glycan complexes containing multiple glycans, capable of reconstructing multivalent interactions and demonstrating strong avidity towards receptors. Various platforms, including polymers, nanoparticles, liposomes, self-assembled materials, oligovalent scaffolds (such as calixarenes, cyclodextrins, and cyclopeptides), and dendrimers, have been employed to achieve multivalency.

Dendrimers, especially, offer uniform assemblies of glycans. We found that histidine facilitates Cu(I)-mediated Huisgen 1,3-dipolar cycloaddition [22, 23]. By incorporating the N^im-benzylhistidine residue into the peptide substrate, we achieved an efficient ‘self-activating’ click reaction between azide and alkyne-containing peptides, yielding an almost quantitative reaction. Using this ‘self-activating’ click reaction [22] (Fig. 21.11), we synthesized diverse glycodendrimers [24,25,26,27,28,29].

Fig. 21.11

Self-activating click reaction

We successfully synthesized glycodendrimers comprising biantennary type N-glycans, encompassing 16 molecules on a polylysine core [24]. The self-activating click reaction proceeded almost quantitatively, and subsequent labeling via 6π azaelectrocyclization afforded PET probes 70a, 71a, 72a and fluorescent probes 70b, 71b, 72b. Employing positron emission tomography (PET) and fluorescence imaging of sialylated and asialylated N-glycan dendrimers, we visualized the sialic acid-dependent circulation and retention in vivo (Fig. 21.12).

Fig. 21.12

Synthesis of N-glycan dendrimers for PET and fluorescence imaging

Multivalency plays a crucial role in pathogen recognition of host cells, facilitating strong adhesion of pathogens to these cells. In the pursuit of developing inhibitors to prevent pathogenic infections, there have been reports of synthesizing numerous glycan clusters exhibiting multivalent effects. Using the self-activating click chemistry method developed by our group, we synthesized antipathogenic glycodendrimers [28]. The remarkable reactivity of this method enabled the efficient preparation of dendrimers containing anti-influenza sialyl trisaccharide 75 (Fig. 21.13) or Gb3 trisaccharide 79 (Fig. 21.14). These dendrimers exhibited strong avidity toward hemagglutinin on the influenza virus and the Shiga toxin B subunit, respectively. These glycodendrimers are anticipated to be effective antipathogenic compounds.

Fig. 21.13

Synthesis of anti-influenza sialyl trisaccharide dendrimers

Fig. 21.14

Synthesis of Gb3 trisaccharide dendrimers

We synthesized 16-mer B-antigen-displaying dendrimers 88, 89, and 90 of various sizes and assessed their interaction with IgM antibodies to explore the critical factors influencing effective multivalency [29]. Surprisingly, even the smallest dendrimer 88, unable to fully occupy IgM’s multiple binding sites, demonstrated distinct multivalent behavior with affinity levels comparable to or surpassing those of larger dendrimers 89 and 90. These findings highlight the significance of the statistical rebinding model, suggesting that the rapid exchange of clustered glycans significantly contributes to glycodendrimers’ multivalent interactions. This indicates that high-density glycan presentation for enhanced statistical rebinding is crucial for multivalent interaction. This contrasts with the prevailing emphasis on the chelation model. Consequently, our study offers novel insights and essential guidelines for crafting glycodendrimers at a molecular level (Fig. 21.15).

Fig. 21.15

Synthesis of B-antigen-displaying dendrimers

The majority of animals possess α-gal, an antigenic glycan that is absent in old world monkeys, apes, and humans. Instead, these primates possess a substantial amount of natural anti-Gal antibodies against α-gal. Consequently, α-gal can trigger intense immune reactions in these primates. We engineered a conjugation of α-gal 91 with anti-tumor antibody anti-CD20 or its half-antibody (hAb) [27]. These conjugated antibodies recognized cancer cells, recruiting anti-Gal antibodies to these cells, thereby initiating an additional immune response from the anti-Gal antibodies. α-Gal 91 and dendrimerized α-gal 92 and 93 were conjugated with the hAb to obtain α-gal-hAb conjugates 94, 95, and 96. While the hAb exhibited almost no complement-dependent cytotoxicity (CDC), the α-gal-hAb conjugates exhibited stronger CDC, dependent on the amount of introduced glycans (Fig. 21.16). This approach shows promise in reducing antibody dosages and revitalizing antibodies with insufficient activity.

Fig. 21.16

Synthesis of conjugates of α-gal dendrimers with half-antibody and their complement-dependent cytotoxicity

21.5 Conclusion

The chemical synthesis of A. faecalis lipid A has unveiled its capacity to modulate immune signals; it efficiently activates the immune system without triggering excessive inflammation, and effectively induce IgA for mucosal immunity and IgG for systemic immunity, making it an exceptional vaccine adjuvant.

Sialic acid-containing N-glycans were synthesized successfully using the diacetyl strategy. Our study, employing asymmetrically deuterium-labeled biantennary N-glycans, revealed the H1N1 neuraminidase’s preference for cleaving the sialic acid residue in the α1,3 branch of the biantennary N-glycan. We have been advancing the mechanistic analysis of immune regulation by N-glycans [30,31,32,33].

We successfully synthesized glycan dendrimers using self-activating click reactions. The complexes formed between the natural antibody ligand, α-gal dendrimers, and the anti-tumor antibody CD20 exhibited significant complement-dependent cytotoxicity (CDC) activity.

In conclusion, our endeavors in chemical synthesis have resulted in the creation of molecules capable of modulating the immune system.