Synthesis of ABO blood group antigens and functional glycan display on the cell surface

ABO blood group antigens are involved in various biological phenomena, including immune responses and infections. We achieved efficient and scalable synthesis of A, B, and H antigens. Using chemical conjugation, synthesized B antigen was displayed on the surface of living cells and its function as an antigen was confirmed by the IgM antibody recognition. Our results indicate that the multivalent interactions induced by cell surface glycan clustering are crucial in this system. The prepared cells displaying the glycan antigen are expected to be a model cell for investigating ABO antigen function


Introduction
2][3] Glycans cover the cell surface as a glycocalyx, consisting of glycoproteins and glycolipids, and provide the milieu for the first contact with the external environment.Therefore, glycans are closely related to self-recognition and non-self-recognition, such as infections and immune responses.On the other hand, the structural diversity and heterogeneity of glycans make it difficult to elucidate their functions at the molecular level.Thus, chemical synthesis, which provides a certain amount of pure glycans, is a powerful tool for investigating of the biological functions of glycans.
ABO blood types are categorized by the glycan structure on erythrocytes (Figure 1a).Individuals of O, A, and B blood types express H, A, and B antigens, respectively, with H antigen is a precursor of A and B antigens.ABO blood group glycans act as antigens and individuals produce antibodies, mainly IgM antibodies, against the glycans they do not express.The interaction of ABO blood antigens with their antibodies results in blood agglutination. 4Such immune responses are extremely important in considering blood transfusions.In addition, ABO blood group glycans are expressed on a wide range of organs, including epithelium, intestine, gastric, and pancreas, and thereby, are closely associated to various diseases. 510][11][12][13][14][15][16][17][18][19] Figure 1.a) Chemical structure of ABO blood group glycans.b) Preparation schematic of cells displaying synthetic antigen glycan using chemical conjugation.
Cell surface glycan engineering is a powerful tool for investigating and revealing glycan functions. 20enetic approaches, including the knockout or knockdown of glycosyltransferase, are the most classical and versatile methods and have played a pivotal role in elucidating glycan functions.Bertozzi et al. developed metabolic labeling by incorporating unnatural monosaccharide analogs having the reaction handle followed by the bioorthogonal reaction. 21This method provides a facile platform for installing new chemical functionality to glycans.Chemical [22][23] and chemoenzymatic [24][25][26][27] glycan engineering have also been reported.9][30][31][32][33] Henry et al. reported A and B antigen display on erythrocytes using synthetic glycolipids. 29Palcic et al. reported B antigen introduction onto cell surface by enzymatic labeling. 34n the current study, we synthesized ABO blood group glycans and investigated the display of the synthesized glycans on the cell surface.We achieved efficient and scalable synthesis of ABO blood group glycans.Synthesized B antigen was introduced onto the surface of living cells by chemical conjugation using Nhydroxysuccinimide (NHS) ester (Figure 1b).This approach allowed the display of homogeneous synthetic glycans on the cell surface with easy operation.Furthermore, B antigen displayed by this method was recognized by IgM antibody specific for B antigen glycan.In contrast, the IgM antibody did not interact with B antigen introduced onto the IgG antibody.These results indicate that multivalent interactions through cell surface glycan clustering are crucial for the function of B antigen glycans as an antigen.The prepared cells displaying antigen glycan is expected to be a useful model for investigating ABO antigen function.

Results and Discussion
The synthetic plan of H, A, and B antigens is shown in Scheme 1.We synthesized ABO blood group type II antigens, which are widely expressed in the human body.We initiated the synthesis from galactose derivative 1, which had the orthogonal protecting groups (tert-butyl(dimethyl)silyl (TBS) and fluorenylmethoxycarbonyl protecting group (Fmoc)) at the glycan elongation sites.8, 19 In this study, orthogonally protected galactose fragment 1 was utilized to enable the straightforward synthesis of ABO blood group antigens.-Selective galactosylation between 1 and 2 using neighboring group participation of Fmoc group yielded disaccharide 3.After cleavage of Fmoc group, -fucosylation with 4 produced trisaccharide 5. Trisaccharide 5 can be used as a common intermediate; H-antigen can be obtained by deprotection, whereas A and B antigens can be obtained by cleavage of TBS group followed by galactosaminylation and -galactosylation, respectively.The allyl group at the reducing termini of 5, 8, and 9 can be used as handles for bioconjugation.

Scheme 1. Synthetic strategy of H, A, and B antigens.
Synthesis of H antigen 16 is shown in Scheme 2. Compound 10, reported previously by our group, 35 was converted to a galactosyl donor 1. Selective cleavage of benzylidene 36 followed by benzoyl protection generated 1. Glycosylation between 1 and 2 37 using NIS and TfOH 38 as activators afforded 3 with perfect selectivity due to neighboring group participation.In all case in this study, the stereochemistries produced by glycosylations were determined by the coupling constants at the anomeric positions.Cleavage of Fmoc group resulted 12, which was then fucosylated with 4 39 to produce 5 in 88% yield with perfect -selectivity.
With the common intermediate 5 in hand, H antigen 16 was synthesized via deprotection and introduction of the carboxylic acid for bioconjugation.After cleavage of TBS with HF•pyridine, Troc group of 13 was converted to acetamide through the reduction of azide followed by acetylation.In this step, the 3 position of galactose was also acetylated.A carboxy methyl group was then introduced to the reducing terminal of 14 by olefin metathesis reaction with methyl acrylate to yield 15. Global deprotection of 15 by hydrogenation and hydrolysis afforded H antigen 16.

Scheme 2. Synthesis of H antigen 15.
A antigen 18 and B antigen 20 were synthesized from 13 (Scheme 3).Glycosylation with 2-azido galactosaminyl donor 6 40 using AgClO4 41 gave protected A antigen 17 in 90% with perfect -selectivity.Synthesis of A antigen 18 was then completed via introduction of carboxylic acid and deprotection, as was done in the synthesis of H antigen 15.B antigen 20 was similarly synthesized.After -selective galactosylation with 7, 42 the resulting 19 was converted to B antigen 20.Scheme 3. Synthesis of A antigen 18 and B antigen 20.

AUTHOR(S)
With ABO blood group antigens in hand, we investigated the introduction of synthetic glycan onto the cell surface (Figure 2).We herein used B-cell lymphoma Raji cells and B antigen.We employed direct chemical conjugation of glycan onto the cell surface proteins using NHS ester, as well as indirect labeling using glycanantibody conjugates (Figure 2c).In the indirect method, anti-CD20 antibody, which is used for the treatment of B-cell lymphoma, was applied as Raji cell recognition antibody.2a).Reaction of anti-CD20 antibody with two concentrations of 21 (0, 1.0, and 5.0 mM) in PBS at 4 °C for 30 min afforded the antibodies conjugated with various loading ratios of B antigen (Figure 2b).Based on SDS-PAGE analysis, the loading ratio of B antigen increased as the concentration of 21 was increased.The average loading ratios (n) were estimated to be 7 and 12 when 1.0 and 5.0 mM 21 was used, respectively.
After labeling Raji cell with B antigen using B antigen NHS ester 21 or B antigen-antibody conjugate, the interaction with IgM antibody specific for B antigen glycan was determined using flow cytometry to evaluate the function of the B antigen displayed on the cell surface (Figure 2c).The treatment of Raji cell with 5.0 mM 21 in PBS at 37 °C for 30 min did not decrease cell viability, and the treated cells were successfully recognized by the fluorescein-labeled anti-B antigen IgM antibody.In contrast, cells treated with B antigen-antibody conjugates were not recognized by the IgM antibody.Considering the successful staining with the fluoresceinlabeled anti-mouse IgG antibody, these results showed that B antigen loaded on anti-CD20 antibody was not recognized by the IgM antibody.In our previous report using -gal as a glycan antigen, we demonstrated -gal conjugated to anti-CD20 antibody in the same manner described in the current study was recognized by antibodies against -gal, 43 suggesting that accessibility of the glycan antigen on anti-CD20 antibody does not the issue.The difference in recognition of B antigen and -gal might be attributed to the binding affinity of the corresponding antibodies as antibodies against -gal contain a certain amount of IgG antibodies, which exhibit high affinity.
The results described above indicate that the method used for glycan displaying is important for synthetic glycans to exhibit their function on the cell surface.5] Such multivalency plays a crucial role in glycan recognition.7][48] In the presence of a multivalent binding partner, membrane proteins can diffuse in lipid bilayer to form clusters.0] When B antigen was introduced to the membrane proteins using NHS ester 21, the clustering mechanism was expected to enhance the interaction between B antigen displayed on the cell surface and IgM antibody.However, B antigen introduced onto the anti-CD20 antibody was not flexible, and thereby, may not provide multivalent interaction with IgM antibody.Multivalent interaction by CD20 clustering might be prevented by steric hindrance of anti-CD20 antibody.These results demonstrated that reconstruction of multivalent interaction is crucial for the functional display of synthetic glycans in biological systems.ABO blood group glycans are known to primarily exist on glycolipids and are also expressed on glycoproteins as N-glycans and O-glycans.However, their recognition on glycoproteins by the IgM antibodies has not been elucidated.Glycan display using NHS ester 21 is usually directed against amines on proteins to produce pseudo-glycoproteins.Thus, the current study suggests that ABO blood group glycans on glycoproteins can also be recognized by IgM antibodies through their clustering and can induce biological events.

Conclusions
We synthesized ABO blood group glycans and achieved functional display of B antigen on the surface of a living cells.Importantly, our synthesis was efficient and scalable.Orthogonal protection strategy enabled the straightforward synthesis of ABO blood group antigens.All glycosylations proceeded in more than 70% yield with perfect stereoselectivity and each glycan (16, 18, and 20) was procured at quantities greater than 50 mg.Synthesized B antigen was displayed on the cell surface in two ways, direct chemical conjugation using NHS ester 21 and indirect labeling using the glycan-antibody conjugate.B antigen directly conjugated onto cell surface proteins was recognized by IgM antibody, whereas B antigen conjugated onto IgG antibody was not recognized.These results indicate that the multivalent interactions induced by glycan clustering are important for the emergence of proper glycan function on the cell surface.Our future work will include using this newly developed approach for displaying "functional" synthetic glycan antigen on the cell surface to elucidate ABO antigen function in various biological phenomena, including immune responses and pathogen infections.

Experimental Section
General. 1 H and 13 C NMR spectra were recorded in an indicated solvent with JEOL ECA 500 MHz spectrometer.Chemical shifts of 1 H and 13 C NMR were referenced to the solvent peaks: δ=7.26 and δ=77.16 for CDCl3, δ=3.30 and δ=49.30for CD3OD.Multiplicities abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad.High-resolution mass spectra were recorded on a ESI-LTQ-Orbitrap XL (FTMS) mass spectrometer and ESI-Q-TOF mass spectrometer.Chemical purification was carried out using silica-gel column chromatography.Silica-gel column chromatography was carried out using Silica Gel 60N (Kanto Chemical Co., 40-50 µm or 63-210 µm) at medium pressure (1-4 kg cm -2 ).Gel permeation chromatography was carried out using Sephadex LH-20 at atmospheric pressure.Silica-gel 60 F254 (Merck Co.) was used for TLC analysis and preparative TLC purification, and compounds were visualized by UV (254 nm), p-methoxybenzaldehyde (panisaldehyde, 0.03% in EtOH-H2SO4-acetic acid buffer).Anhydrous CH2Cl2 were distilled in the presence of calcium hydride.Anhydrous THF, DMF, distilled water, and toluene were purchased from FUJIFILM Wako Pure Chemical Corporation, Ltd.. Nonaqueous reactions were carried out under argon atmosphere.Molecular sieve 4A (MS4A) was activated with a microwave and dried in vacuo for 3 times before use.All other commercially available reagents and solvents were used as purchased.

Figure 2 .
Figure 2. a) Preparation of B antigen NHS ester 20.b) Preparation of B antigen-antibody conjugate.c) B antigen labeling of cell surfaces and evaluation of the labeled cells using anti-B antigen IgM antibody.

Figure 3 .
Figure 3. Putative interaction of B antigen with IgM antibody.a) B antigen (red circle) chemically conjugated to the cell surface membrane protein.b) B antigen chemically conjugated to IgG antibody.