A New Chemical Approach to Human ABO Histo-Blood Group Type 2 Antigens

A new chemical approach to synthesizing human ABO histo-blood type 2 antigenic determinants was developed. N-Phthaloyl-protected lactosaminyl thioglycoside derived from lactulose via the Heyns rearrangement was employed to obtain a type 2 core disaccharide. Use of this scheme lowered the overall number of reaction steps. Stereoselective construction of the α-galactosaminide/galactoside found in A- and B-antigens, respectively, was achieved by using a unique di-tert-butylsilylene-directed α-glycosylation method. The proposed synthetic scheme provides an alternative to existing procedures for preparing ABO blood group antigens.


Introduction
ABO histo-blood group antigens are expressed on red blood cells and are widely distributed in various tissues such as the vascular endothelium, where they are displayed on plasmalemmal glycoproteins and glycolipids by attachment to sugar residues that terminate N-linked, O-linked, and lipid-linked glycans [1,2]. The A, B, and O group antigens are defined by the GalNAcα (1)(2)(3)[Fucα(1-

Results and Discussion
The typical procedure for synthesizing ABO blood group antigens is stepwise assembly of the monosaccharide unit, which requires a laborious protection/deprotection strategy for the multistep preparation of both monosaccharide donor and acceptor. To improve accessibility to those antigens, we designed a unique synthetic route to the target ABO group type 2 antigenic oligosaccharides. As shown in Scheme 1, our synthetic strategy involves two key reactions: (1) the Heyns rearrangement for simple preparation of N-acetyl-lactosamine (4-O-β-D-galactopyranosyl-D-N-acetyl-glucosamine), a  (2) di-tert-butylsilylene (DTBS)-directed α-galactosaminylation and α-galactosylation for the formation of A and B determinants, respectively.

Scheme 1. Retrosynthetic analysis of target compounds.
The Heyns rearrangement is known to be effective for obtaining a lactosamine derivative by simple manipulation starting from lactulose (4-O-β-D-galactopyranosyl-D-fructose). This reaction was originally developed for converting ketoses into the corresponding 2-amino-2-deoxyaldoses [32]. We hoped that the use of lactulose (4) as an alternate starting material would allow us to minimize the number of reaction steps as well as to reduce the time and effort needed. Additionally, lactulose is a relatively inexpensive and commercially available sugar. Recently, Wrodnigg and co-workers reported an improved Heyns rearrangement procedure, which was much more practical than the original procedure [33,34]. Other groups have recently reported even more practical protocols suitable for large-scale synthesis [35,36]. In the present study, we followed these procedures to obtain lactosamine derivative 5 [37] as a key building block. Compound 5 was efficiently prepared in five steps (Scheme 2) by methods in the literature [33][34][35][36]. Conversion of peracetate derivative 5 into thioglycoside form was performed in the presence of ethanethiol and BF 3 ·OEt 2 in 1,2-dichloroethane to give ethylthioglycoside 6 in 96% yield. The ethylsulfinyl group was selected in consideration of its solubility in MeOH, which was used in the next step. A phenylsulfinyl group in place of the ethylsulfinyl group resulted in poor solubility in MeOH, leading to a poor results in the deacetylation reaction. After removal of all acetyl groups in 6, hydroxyl groups at the C2 and C3 positions of the galactose residue were simultaneously protected as a butanediacetal (BDA) [38] to afford compound 8. In this reaction, a regioisomer of 8, namely, a 3,4-O-BDA-protected by-product, was formed and these regioisomers were separated by silica gel column chromatography. However, small amounts of impurities could not be separated from 8. Acetylation of 8 along with contaminants and subsequent hydrolysis of the BDA group afforded diol 10 as the sole product in 57% yield over the four operations. The tin-mediated selective acylation developed by Muramatsu [39] was then applied to selectively protect the C3′-OH group by the Troc group, giving the disaccharide acceptor 11 in 84% yield. Another procedure for selective protection of the C3′-OH group by treatment of TrocCl with pyridine in CH 2 Cl 2 at lower temperature (−40 °C) gave 11 in somewhat lower yield (76%). For next glycosylation, the fucosyl N-phenyltrifluoroacetimidate 12 was designed to increase both reactivity and stability as a fucose donor. The previously used fucosyl donor, 2,3,4-tri-O-benzyl-protected fucosyl imidate, could be served as a good fucosyl donor, but was relatively unstable under glycosylation conditions due to its armed feature. Chemo-selectively removable PMB group was chosen as a protecting group at C2 position and electron-withdrawing acetyl groups at C3 and C4 were incorporated to suppress the armed feature by the PMB group, which could lead to stabilization of the donor. Furthermore, a more stable N-phenyltrifluoroacetimidate group compared to a trichloroacetimidate group was used as a leaving group [40,41]. The glycosylation of 11 with 12, which was derived from a known fucose derivative [42] and was promoted by TMSOTf in a mixed solvent system of cyclopentylmethyl ether (CPME)-dichloromethane (1:1) [43] at −80 °C, provided trisaccharide 13. Small amounts of contaminates remained after column chromatography. The mixture containing contaminants was used directly in the next reaction. Removal of the p-methoxybenzyl (PMB) group under acidic conditions allowed for purification of the newly formed trisaccharide, affording 14 with a yield of 88% over two steps. Acetylation of the liberated hydroxyl group afforded compound 15 with a yield of 95%. Next, the coupling reaction of 15 with N-Cbz-protected aminopentanol 16 occurred smoothly in the presence of N-iodosuccinimide (NIS) and TfOH [44,45] in CH 2 Cl 2 at 0 °C to give the desired glycoside 17 in 85% yield. Subsequent deprotection of the Troc group by treatment with zinc and AcOH [46] in 1,2-dichloroethane at 40 °C afforded common trisaccharide derivative 18 with a yield of 90%.
For constructing the A and B antigen skeletons, it is necessary to incorporate galactosamine (for A antigen) and galactose (for B antigen) residues into trisaccharide 18 in α-linked form. Typically, α-D-galactosides are obtained by using ethereal solvents such as diethyl ether and 1,4-dioxane as well as the anomeric effect [47]. Scheme 2. Synthesis of the common trisaccharide unit.
However, highly α-selectivity in such galactosylation is generally difficult and strongly dependent on various factors, such as the substrate structure, promoter, and temperature. The stereoisomers formed are often difficult to separate, which presents a serious disadvantage for synthetic studies. In 2003, we developed a reliable method for α-selective galactosidation andgalactosaminidation using DTBS-protected glycosyl donors [48][49][50][51]. Notable features of the DTBS-directed α-galactosylation are excellent α-selectivity even in the presence of a neighboring participating group on the C2 oxygen or nitrogen, and the relatively greater difference between the R f values of the α and β isomers that enables them to be more easily separated. Thus, we decided to utilize DTBS-directed α-galactosylation for the construction of the A and B antigen sequences.
As shown in Scheme 3, trisaccharide acceptor 18 was glycosylated with galactosaminyl donor 19 [48] and galactosyl donor 20 [52] in the presence of NIS and TfOH in CH 2 Cl 2 at 0 °C, giving the corresponding tetrasaccharides 21 and 22 in α-linked form in yields of 82% and 58%, respectively. In these reactions, the recovery of unreacted acceptor 18 was 9% and 22%, when 19 and 20 were used, respectively, despite the use of 2 equiv of donor. However, other possible stereoisomers were not detected and both α-products were easy to isolate by column chromatography. To our surprise, the coupling yield of 22 was moderate. When we attempted to use the armed 2,3-di-O-benzyl-type galactose donor instead of 20, the yield was not improved (41%) and many unidentified by-products were generated. The unexpectedly low reactivity of 18 as a glycosyl acceptor might arise from steric hindrance around 3-OH on the Gal residue. On the route to the target compounds, there is a global deprotection sequence (Scheme 4). Selective removal of the Troc groups of 21 by treatment with zinc and AcOH, followed by selective acetylation of the liberated amine of the galactosamine residue at C2 afforded 23 in 84% yield. Then, removal of the DTBS group with tributylamine hydrofluoride (TBAHF) in THF [53] followed by acetylation of the hydroxyl groups provided 24 in 98% yield over two steps. After removal of all acetyl groups on 24, the phthalimide group at C2 of the glucosamine residue was converted to an acetamide group by sequential treatment with hydrazine hydrate in refluxing EtOH followed by selective acetylation of the free amine, affording 25 in 80% yield over three steps. Finally, the Cbz group at the terminus of the linker was removed by hydrogenolysis with Pd/C under hydrogen atmosphere, thus furnishing target 1 (A antigen) in 81% yield. Similarly, the deprotection of compounds 22 and 18 were efficiently carried out to furnish target compounds 2 (B antigen) and 3 (O antigen) in good yields.

General Methods
All reactions were carried out under a positive pressure of argon, unless otherwise noted. All chemicals were purchased from commercial suppliers and used without further purification, unless otherwise noted. Molecular sieves were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and dried at 300 °C for 12 h in a muffle furnace prior to use. Solvents as reaction media such as CH 2 Cl 2 , MeOH, THF, DMF, and pyridine, which were tapped off from The Solvent Supply System, were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and used without purification. TLC analysis was performed on Merck TLC (silica gel 60F254 on glass plate, Darmstadt, Germany). Compound detection was either by exposure to UV light (2536 Å) or by soak in a solution of 10% H 2 SO 4 in ethanol followed by heating. Silica gel (80 mesh and 300 mesh) manufactured by Fuji Silysia Chemical Ltd. (Kasugai, Japan) was used for flash column chromatography. Quantity of silica gel was usually estimated as 100 to 200-fold weight of sample to be charged. Solvent systems in chromatography were specified in v/v. Evaporation and concentration were carried out in vacuo. 1 H-NMR and 13 C-NMR spectra were recorded with Bruker Biospin AVANCE III 500/800 spectrometers (Billerica, MA, USA). Chemical shifts in 1 H-NMR spectra are expressed in ppm (δ) relative to the signal of Me 4 Si, adjusted to δ 0.00 ppm. Data are presented as follow: Chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = double of doublet, td = triple doublet, m = multiplet and/or multiple resonances), integration, coupling constant in Hertz (Hz), position of the corresponding proton. COSY methods were used to confirm the NMR peak assignments. High-resolution mass (ESI-TOF MS) spectra were run in a Bruker Daltonics micrOTOF (Billerica, MA, USA). Optical rotations were measured with a 'Horiba SEPA-300' high-sensitive polarimeter (Kyoto, Japan).  (6). To a mixture of 5 (4.16 g, 5.44 mmol) in (CH 2 Cl) 2 (27.2 mL) were added EtSH (606 µL, 8.16 mmol) and BF 3 ·OEt 2 (1.03 mL, 8.16 mmol) at 0 °C. After stirring for 2 h at rt as the reaction was monitored by TLC (3:2 EtOAc-hexane), the reaction was quenched by the addition of crushed ice. The solution was diluted with CHCl 3 and subsequently washed with ice-cooled H 2 O, satd aq Na 2 CO 3 , and brine. The organic layer was then dried over Na 2 SO 4 , and concentrated. The resulting residue was purified by silica gel column chromatography (1:1 EtOAc-hexane) to give 6 (3.99 g, 96%). Spectroscopic data of 6 were identical to those reported in the literature [54]. (10). To a solution of 6 (1.08 g, 1.41 mmol) in MeOH/CH 2 Cl 2 (2:1, 14.1 mL) was added NaOMe (28% solution in MeOH, 31.9 µL, 141 µmol) at 0 °C. After stirring for 2 h at rt as the reaction was monitored by TLC (3:2 EtOAc-hexane), the reaction was neutralized with AcOH. After concentration, the resulting residue was diluted with CHCl 3 and subsequently washed with H 2 O and brine. The organic layer was dried over Na 2 SO 4 , of which solid was filtered through cotton and the filtrate was then evaporated (giving 7). The residue was subjected to next reaction without further purification. The crude product 7 was dissolved in MeOH (28.2 mL). To the solution were added 2,3-butanedione (492 µL, 5.64 mmol), trimethyl orthoformate (1.95 mL, 17.8 mmol), and (±)-10-camphorsulfonic acid (66 mg, 282 µmol) at rt. After stirring for 20 h at reflux as the reaction was monitored by TLC (10:1 CHCl 3 -MeOH), the reaction was quenched by the addition of triethylamine (218 µmol) and concentrated. The resulting residue was diluted with CHCl 3 and subsequently washed with H 2 O and brine. The organic layer was dried over Na 2 SO 4 , filtered, concentrated. The resulting residue was roughly purified by silica gel column chromatography (20:1 CHCl 3 -MeOH) to give 2,3-O-BDA-protected product 8 along with small amounts of contaminants. The crude mixture (494 mg) was dissolved in pyridine (7.8 mL). To the solution were added Ac 2 O (890 µL, 9.42 mmol) and a catalytic amount of DMAP at 0 °C. After stirring for 1 h at rt as the reaction was monitored by TLC (3:2 EtOAc-hexane), the mixture was co-evaporated with toluene. The resulting residue was diluted with EtOAc and subsequently washed with 2 M HCl, H 2 O, satd aq NaHCO 3 , and brine, dried over Na 2 SO 4 , and concentrated. The resulting residue was purified by silica gel column chromatography (2:3 EtOAc-hexane) to give 9 (634 mg), to which suspension in H 2 O (1.6 mL) was added trifluoroacetic acid (14.4 mL) at 0 °C. After stirring for 2 h at rt as the reaction was monitored by TLC (1:1 CHCl 3 -acetone), the mixture was diluted with toluene and concentrated. The resulting residue was purified by silica gel column chromatography (7:3 CHCl 3 -acetone) to give 10 (548 mg, 57% over four steps).  Ethyl [4,6- (15). To a solution of 14 (1.06 g, 975 µmol) in pyridine (4.9 mL) was added acetic anhydride (4.9 mL) at 0 °C. After stirring for 2 h at rt as the reaction was monitored by TLC  (17 (18 170.1, 170.1, 169.9, 156.2, 136.6, 134.3, 131.4, 128.5, 128.0, 123.5, 100.1, 98.1, 97.8, 74.9, 73.0, 72.4,  71.1, 71.0, 69.9, 69.7, 69.6, 68.2, 67.7, 66.5, 65.2, 62.4, 61. 5, 54.8, 40.8, 29.6, 29.3, 28.8, 23.0, 20.8, 20.7, 20.6, 20.6, 20.5, 15.7 (20). To a solution of Phenyl 3-O-benzyl-1-thio-β-D-galactopyranoside (262 mg, 724 µmol) in pyridine (7.2 mL) was added di-tert-butylsilyl bis(trifluoromethanesulfonate) (260 µL, 796 µmol) at 0 °C. After stirring for 3.5 h at 0 °C as the reaction was monitored by TLC (1:1 EtOAc-hexane), benzoic anhydride (328 mg, 1.45 mmol) was added to the mixture at 0 °C. After stirring for 22 h at rt as the reaction was monitored by TLC (1:3 EtOAc-hexane), the reaction was quenched by the addition of MeOH at 0 °C. The mixture was co-evaporated with toluene. The residue obtained was diluted with EtOAc, washed with 2 M HCl, H 2 O, satd aq NaHCO 3 , and brine, dried over Na 2 SO 4 , concentrated. The resulting residue was purified by silica gel column chromatography (1:7 EtOAc-hexane) to give 20 (324 mg, 74%).   (21). A mixture of 18 (103 mg, 91.1 µmol) and 19 (138 mg, 182 µmol), and NIS (46 mg, 364 µmol) was exposed to high vacuum for 1 h. The mixture was dissolved in CH 2 Cl 2 (2.7 mL), to which 4 Å molecular sieves (273 mg) was added at rt. After stirring for 30 min at rt and then for 10 min at 0 °C, TfOH (1.9 µL, 18.2 µmol) was added to the mixture. After stirring for 3 h at 0 °C as the reaction was monitored by TLC (3:1 EtOAc-hexane, 1:1 EtOAc-hexane, 1:3 EtOAc-hexane), additional portions of NIS (23 mg) and TfOH (1.0 µL) were added to the mixture and the stirring was continued. After stirring for total 5 h, the reaction was quenched by the addition of satd aq NaHCO 3 . The precipitate was filtered through Celite. The filtrate was diluted with CHCl 3 , washed with satd aq Na 2 S 2 O 3 and brine. The organic layer was subsequently dried over Na 2 SO 4 , concentrated and the residue was then purified by silica gel column chromatography (1:1 EtOAc-hexane) to give 21 (132 mg, 82%), and 9.5 mg (9%) of 18 was recovered.   After stirring for 1.5 h at 0 °C as the reaction was monitored by TLC (3:1 EtOAc-hexane, 1:1 EtOAc-hexane, 1:3 EtOAc-hexane), additional portion of TfOH (1.0 µL) was added to the mixture and the stirring was continued. After stirring for total 2 h, the reaction was quenched by the addition of satd aq NaHCO 3 . The precipitate was filtered through Celite. The filtrate was diluted with CHCl 3 , washed with satd aq Na 2 S 2 O 3 and brine. The organic layer was subsequently dried over Na 2 SO 4 , concentrated and the residue was then purified by silica gel column chromatography ( (23). To a solution of 21 ( (24). To a solution of 23 ( (25). To a solution of 24 (19.8 mg, 13.6 µmol) in MeOH (1.4 mL) was added NaOMe (1M solution in MeOH, 6.8 µL, 6.78 µmol) at 0 °C. After stirring for 4 h at rt as the reaction was monitored by TLC (20:12:1 CHCl 3 -MeOH-H 2 O), the reaction was neutralized with Muromac (H + ) resin. The resin was filtered out and the filtrate was concentrated. The residue obtained was then dissolved in EtOH (2.8 mL). To the solution was added NH 2 NH 2 ·H 2 O (1.0 µL, 27.2 µmol) at rt. The reaction mixture was stirred at reflux as monitored by TLC (5:4:1 CHCl 3 -MeOH-H 2 O). Additional portions of NH 2 NH 2 ·H 2 O (2.0 µL) was added to the mixture every 15 min (total amounts of NH 2 NH 2 ·H 2 O added was 32 µL). After 6.5 h, the reaction mixture was concentrated and exposed to high vacuum for 1 h. The resulting residue was then dissolved in MeOH/CH 2 Cl 2 (3:1, 4.4 mL). To the mixture was added acetic anhydride (26 µL, 272 µmol) at 0 °C. After stirring for 1.5 h at rt as the reaction was monitored by TLC (5:4:1 CHCl 3 -MeOH-H 2 O), the reaction mixture was concentrated. The residue obtained was purified by silica gel column chromatography (Iatrobeads, 9:5:0.

Conclusions
We have developed a novel approach to synthesizing human histo-blood group type 2 antigens. A lactosamine derivative served as a key building block and was efficiently prepared from lactulose via the Heyns rearrangement, a strategy that allowed us to lower the overall number of reaction steps. The introduction of galactosamine and galactose in α-linked form into the O-antigen trisaccharide was accomplished by a unique DTBS-directed α-glycosylation to afford type 2 A-and B-antigen tetrasaccharides, respectively. The present synthetic protocol can provide rapid access to various biologically relevant glycoconjugates that contain N-acetyl-lactosamine and ABO blood group antigens. Studies on biological applications using the synthesized antigens will be reported in due course.