The Synthesis of Blood Group Antigenic A Trisaccharide and Its Biotinylated Derivative

Blood group antigenic A trisaccharide represents the terminal residue of all A blood group antigens and plays a key role in blood cell recognition and blood group compatibility. Herein, we describe the synthesis of the spacered A trisaccharide by means of an assembly scheme that employs in its most complex step the recently proposed glycosyl donor of the 2-azido-2-deoxy-selenogalactoside type, bearing stereocontrolling 3-O-benzoyl and 4,6-O-(di-tert-butylsilylene)-protecting groups. Its application provided efficient and stereoselective formation of the required α-glycosylation product, which was then deprotected and subjected to spacer biotinylation to give both target products, which are in demand for biochemical studies.


Introduction
Since the discovery of the ABO blood group system and the role of carbohydrate residues in blood antigens [1,2], there has been continued interest in developing new synthetic approaches to the assembly of carbohydrate blood group antigen determinants [3][4][5][6][7][8][9][10]. Besides playing an important role in blood cell recognition and blood group compatibility, blood transfusion, and organ transplantation [11][12][13], A trisaccharide and structurally related compounds can be used as haptens to test the carbohydrate specificities of plant [14] and mammalian lectins [15,16] and serve as a model for conformational and spectral studies [17] of vicinally branched oligosaccharides. A trisaccharide derivatives can also serve as model compounds in the development of new biomedical technologies, since antibodies against this carbohydrate antigen are commercially available.
A trisaccharide represents the minimal terminal fragment of all blood group A antigens. It has a branched structure where the central β-Gal residue is glycosylated with α-fucose at O-2 and with α-galactosamine at O-3 (see Figure 1). Despite numerous works devoted to the synthesis of oligosaccharides related to blood group antigens, there are only a few papers dedicated specifically to the synthesis of A trisaccharide derivatives [4][5][6]9]. Herein, we report on the assembly of spacered A trisaccharide 1a and its biotinylated derivative 1b, making use of the new galactose block 4, which bears a set of convenient temporary protecting groups permitting selective liberation of HO-groups as well fucosyl donor 5 [18,19] and bicyclic 2-azido-2-deoxy-selenogalactoside 10 [20] containing stereocontrolling O-protecting groups, which favor the required α-(1,2-cis)-glycosylation. A bulky 4,6-O-(di-tert-butylsilylene)-protecting group at O-4 and O-6 was used to prevent the formation of undesirable β-glycosylation products [21] while a 3-O-benzoyl group was introduced to provide α-stereocontrol through remote anchimeric participation [22].
Donor 10 was recently proposed [20,23] and has been successfully applied in the syntheses of complex linear oligosaccharides related to bacterial and fungal antigenic polysaccharides (see a mini-review [23] and references therein). At the same time, there is still little published data on the glycosylation of vicinally branched oligosaccharides by means of 2-azido-2-deoxy-selenogalactoside donors as well as the application of the donors in the regioselective glycosylation of diolic acceptors. The studies discussed below were planned to fill these gaps.

Results and Discussion
Key steps in the synthesis of spacered A trisaccharide 1a were the regio-and stereoselective building of three glycosidic bonds. While the formation of a β-glycoside bond is a straightforward task, α-glycosylation by fucosyl and galactosamine donors requires the careful selection of protective groups and experimental conditions effective for α-glycoside bond formation [24]. To promote the desired stereoselectivity, a new type of galactosyl acceptor 4 was easily synthesized from tetraol 2 [25] through the introduction of 3,4-Oisopropylidene and 6-O-benzoyl groups, which can be selectively removed in the presence of other protecting O-substituents (Scheme 1).

Scheme 1. Synthesis of galactosyl acceptor 4.
To form the fucosyl block, donor 5 was used. This compound contains two benzoyl protecting groups at O-3 and O-4, which, despite reducing the donor's activity, provide effective α-directing glycosylation stereocontrol through remote anchimeric participation [18,19]. Fucosylation of galactoside 4 proceeded stereoselectively, giving an inseparable mixture of α-isomer 6 and βisomer 7 (Scheme 2) in the ratio~9:1 with a yield of 91%. The anomeric configurations of the Fuc units in disaccharides 6 and 7 were confirmed by the characteristic values of the corresponding C-1 signals in 13 C NMR spectra and J 1,2 constants in the 1H NMR spectra (for 6: 95.5 ppm and 3.4 Hz; for 7: and 103.3 ppm and 8.0 Hz, respectively). The individual α-isomer was purified after the removal of the Oisopropylidene group, which gave the desired diol 8 in a 79% yield. The value of coupling constant J 1,2 (3.6 Hz) confirmed the α-configuration of the Fuc unit in 8. In addition to diol 8, its monohydroxy-derivative 9 was prepared by treatment with trimethyl orthobenzoate [26]  The last step in the assembly of the trisaccharide A backbone was the glycosylation of disaccharide 8 with 2-azido-2-deoxy-galactosyl donor 10, which was prepared via azidophenylselenylation of a triacetylgalactal [23,27] and subsequent selective protection. The glycosylation α-stereoselectivity of donors of this type can be regulated by the reaction solvent [28] and specially selected types of O-protective groups [20][21][22][23]29,30].
It is known that equatorial hydroxyl groups are usually more reactive than axial ones [31,32]. Based on this assumption, we suggest that a regioselective 3-O-glycosylation of diol 8 would be possible. However, the reaction between disaccharide 8 and donor 10 yielded an inseparable mixture of products (Scheme 3). Presumably, it consisted of regioisomers 11 and 12 in a 2.8:1 ratio (NMR data), which were formed via (1→3)-and (1→4)-glycosylation, respectively. To check our assumption, we treated the glycosylation products with BzCl in Py and then removed the 4,6-O-(di-tert-butylsilylene)-protection with HF/Py. As result, we obtained two separate compounds: α-3-Oand α-4-O-glycosylation products 13 and 14, which were identified by NMR spectroscopy. In particular, the formation of α-glycoside bonds was confirmed by coupling constant J 1,2 for the GalN-unit (3.8 and 3.6 Hz in 1 H NMR spectra for 13 and 14, respectively). Regioselectivity of the glycosylation reaction was confirmed by comparing the downfield signals H-3Gal and H-4Gal relative to each other in the 1 H NMR spectra (13: 4.24 ppm for H-3Gal; 5.99 ppm for H-4Gal; 14: 5.42 ppm for H-3Gal; 4.46 ppm for H-4Gal) and by downfield signals C-3Gal of 13 (73.2 ppm) and C-4Gal of 14 (74.5 ppm) in the 13 C NMR spectra.
As an alternative method to conduct 3-O-glycosylation with donor 10, we used monohydroxy-acceptor 9 (Scheme 4). As expected, the coupling of compounds 9 and 10 was stereoselective and gave the desired trisaccharide 15 in an 81% yield, contaminated by traces of isomeric product that was formed due to the migration of a benzoyl group in 9 from O-4 to O-3 in the galactose unit during the reaction. Further removal of the di-tert-butylsilylene-group by HF/Py solution and chromatography purification gave the individual diol 13. The α-configuration of the glycoside bond at the GalN-unit of 13 was confirmed by the characteristic value of the corresponding coupling constant J 1,2 (3.8 Hz) in the 1 H NMR spectrum. Hydrogenolysis of 13 to remove the 2-O-benzyl group at the fucosyl residue and reduce the azide substituent to an amine and subsequent N-acetylation resulted in the formation of trisaccharide 16 in an overall yield of 68%. Its saponification gave the target spacered A trisaccharide 1a (73%), which was then treated with the biotin derivative bearing an activated ester group 17 [33] to give the glycoconjugate 1b. Scheme 4. Assembly of the A trisaccharide backbone and preparation of target compound 1a and its biotinylated derivative 1b.

General Information
All reagents were purchased at Sigma-Aldrich unless otherwise noted. MeCN and CH 2 Cl 2 were distilled over P 2 O 5 and CaH 2 . MeOH was distilled over Mg(OMe) 2 . Anhydrous pyridine and DMF were commercial (Sigma-Aldrich). Molecular sieves AW-300 MS (4Å) were crushed and activated before reaction for 5 min at 400-500 • C in vacuo. Amberlite IR-120 (hydrogen form, Fluka) was washed with 1M aq. HCl, H 2 O, acetone, and dried.
NMR spectra were recorded on Bruker Fourier 300HD (300 MHz), Bruker AV400 (400 MHz), or Bruker AV600 (600 MHz) spectrometers at temperatures denoted on the spectra. The resonance assignment in 1 H and 13 C NMR spectra was performed using 2D-experiments (COSY, HSQC). Chemical shifts are reported in ppm referenced to tetram-ethylsilane as a standard for 1H and solvent signal (δ = 77.16 for CDCl 3 ) for 13 C. 1 H-NMR spectra in D 2 O were registered with water suppression using a presaturation pulse sequence. See all NMR spectra in Supplementary Materials.
High-resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF II instrument using electrospray ionization (ESI). The measurements were performed in positive ion mode (interface capillary voltage −4500 V) or in negative ion mode (3200 V); mass range from m/z 50 to m/z 3000 Da; external or internal calibration was made with an electrospray calibrant solution (Fluka). A syringe injection was used for solutions in a mixture of acetonitrile and water (50:50 v/v, flow rate 3 µL·min −1 ). Nitrogen was applied as a dry gas; interface temperature was set at 180 • C.

3-Aminopropyl 2-Acetamido
To a solution of the trisaccharide 13 (83.8 mg, 66 µmol) in EtOAc (2 mL) and MeOH (1mL) were added 1 M HCl (50 µL) and Pd(OH) 2 /C (100 mg) after the flask was filled with hydrogen. The reaction mixture was stirred for 3 h at RT. Then, the reaction mixture was filtered on a glass filter through a Celite pad and concentrated in vacuo. The crude material was dissolved in CHCl 3 :MeOH (2 mL in ratio 1:1), then Et 3 N (27 µL, 0.19 mmol) and Ac 2 O (12 µL, 0.13 mmol) were added. After completing the reaction, the mixture was concentrated in vacuo and purified by flash chromatography (CHCl 3 :MeOH 0→10%), giving 57.3 mg (68%) of trisaccharide 16. One M MeONa (100 µL) was added to a solution of the purified compound in MeOH:CH 2 Cl 2 (0.8 mL in ratio 3:1). The mixture was left for 2 h, then 4 M NaOH (50 µL) was added and left overnight. The base was neutralized by 1 M aq. HCl and the resulting solution was concentrated. The residue was purified by gel-permeation chromatography (TSK HW-40 (S), 0.1 M AcOH) giving 19.4 mg (73%) of trisaccharide 1a. All NMR and HRMS data corresponded to the literature data [9].

Conclusions
The synthesis of spacered A trisaccharide derivatives 1a and 1b was performed using a 2-azido-2-deoxy-selenogalactoside glycosyl donor bearing stereo-controlling 3-O-benzoyl and 4,6-O-(di-tert-butylsilylene)-protecting groups, showing once again the efficacy of this α-glycosylation agent for the assembly of even vicinally branched oligosaccharide chains. At the same time, we observed rather poor applicability of donor 10 for the regioselective glycosylation of diolic acceptor 8. Obtained trisaccharide 1b is being used in the coating of magnetic nanobeads for glycobiological applications to be described in due course.  Acknowledgments: The authors thank N.E. Ustyuzhanina and D.Z. Vinnitsky for their contribution to the preliminary experiments to this work, D.A. Argunov for recording the NMR spectra and helping with interpreting the data, and also A.I. Tokatly for reading this manuscript and its critical discussion.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of described compounds are available from the corresponding author.