Syntheses of Lewis^X and Dimeric Lewis^X: Construction of Branched Oligosaccharides by a Combination of Pre-activation and Reactivity Based Chemoselective One-Pot Glycosylations

Abstract

Two asymmetrically branched oligosaccharides, Lewis^X and dimeric Lewis^X were assembled in one-pot with high yields and exclusive regio- and stereo-selectivities. p-Tolyl thioglycosides were utilized as the sole type of building blocks, thus simplifying the overall synthetic design. The reactivity independent nature of the pre-activation based method allows modular assembly of the dimeric Lewis^X octasaccharide without the need for tedious protective group manipulation to achieve exact anomeric reactivities.

The Lewis family of oligosaccharides, as represented by Lewis^X pentasaccharide 1 and dimeric Lewis^X octasaccharide 2, is involved in a wide array of biological events, such as modulation of immune system towards a Th2 response,¹ bacterial and viral infection.^2,3 In addition, they are known to be over-expressed on tumor cell surface,^4,5 thus providing a promising target for carbohydrate based anti-cancer vaccine studies.⁶ With their biological significance, structural and stereochemical complexities, Lewis antigens have served as targets for the development of new synthetic methodologies,^7,8 including automated solid phase synthesis,⁹ automated parallel synthesis in solution,⁷ soluble polymer supported synthesis,¹⁰ and reactivity based chemoselective glycosylation.^11,12

Recently, we have developed a pre-activation based chemoselective one-pot glycosylation method, where a thioglycosyl donor is activated in the absence of an acceptor.¹³ Upon completion of the activation, addition of a thioglycosyl acceptor will lead to the formation of a disaccharide containing a thioaryl aglycon, ready for the next round of pre-activation and glycosylation. Multiple glycosylations can be performed in a single reaction flask without intermediate oligosaccharide purifications, thus significantly expediting the glycoassembly process. We have demonstrated that this is a powerful methodology, which has been successfully applied in syntheses of linear oligosaccharides, including hyaluronic acid oligosaccharides,¹⁴ heparin trisaccharides,¹⁵ chitotetraose,¹⁶ and GloboH.¹⁷ Herein, we report the application of the pre-activation based methodology to one-pot construction of branched oligosaccharides.

It is a challenging task to assemble Lewis antigens 1 and 2 in one pot. It is well known that 4-hydroxyl group of glucosamine derivatives has very low nucleophilicity.¹⁸ In addition, fucosylation on 3-OH needs to be carried out with high α selectivity for efficient one pot synthesis. The in situ anomerization procedure for introducing α fucosyl linkage^19,20 is not applicable since the reaction condition cannot be extended to β glycosylations. Furthermore, the rate of glycosylation using in situ anomerization procedure is low requiring room temperature overnight, which is undesirable for multiple sequential glycosylations in one pot. To overcome these difficulties, we designed building blocks 3 to 8 with glucosamine diol 6 serving as a key compound. Keeping C3 hydroxyl group of 6 unprotected reduces steric hinderance to C4 hydroxyl group thus increasing its nucleophilicity. In addition, this allows fucosylation on C3-OH immediately following β-glycosylation of C4-OH without the need to remove C-3 protective group. The N-Phth moiety in 6 is crucial to ensure exclusive regioselectivity for β-galactosylation of 4-OH, as smaller Troc,²¹ azido or acetamido groups²² on C-2 led to regio-isomers. Diol 8 was examined initially as the lactoside acceptor with its axial 4-OH assumed to be much less reactive than the equatorial 3-OH.^10,12,23,24 However, glycosylation of 8 produced two regio-isomeric oligosaccharides in similar quantities.²⁵ The lack of regioselectivity led to the use of lactoside acceptor 7 for our synthesis.²⁶

Pre-activation of galactoside 3 in dichloromethane (DCM) at −78 °C by p-TolSOTf,¹³ formed in situ through reaction of p-TolSCl and AgOTf, was followed by addition of acceptor 6 and a sterically hindered base, tri-tertbutyl-pyrimidine (TTBP)²⁷ (Table 1, entry 1). TLC analysis indicated that acceptor 6 disappeared within just a few minutes. Fucosyl donor 5 was then added to the reaction mixture as a solution in diethyl ether, a solvent known to favor the formation of thermodynamically more stable axial product.^28–30 Because the armed fucosyl donor 5 has high anomeric reactivity,³¹ addition of another equivalent of p-TolSCl chemoselectively activated 5 leading to trisaccharide 10. Lactoside 7 was then added followed by AgOTf and p-TolSCl producing the fully protected Lewis^X pentasaccharide 11 with 40 – 60% yield in just 4 hours for this four-component one pot synthesis.

Table 1.

One-pot Synthesis of Oligosaccharides 10–12, 14.

	Protocol	Donor	B1	B2	B3	Pdt	Yield(%)
1a	A	3	6	5	7	11	40 – 60
2b	B	3	6	5		10	68
3c	B	4	6	5		12	71
4d	B	10	13	7		14	44 – 61

^aDonor:B1:B2:B3 = 1.1: 1.0: 1.1: 0.8

^bDonor:B1:B2 = 1.1: 1.0: 1.1

^cDonor:B1:B2 = 1.4: 1.0: 1.2

^dDonor:B1:B2 = 1.1: 1.0: 0.7

In a separate experiment, the one pot reaction was stopped prior to addition of the lactoside acceptor 7, from which trisaccharide 10 was isolated as the sole trisaccharide in an excellent 68% yield (Table 1, entry 2). Trisaccharide 10 was characterized by extensive NMR experiments. Correlations of anomeric carbon of Gal unit (99.8 ppm) with H₄ of GlcN (4.25 ppm) and anomeric carbon of Fuc (98.0 ppm) with H₃ of GlcN (4.68 ppm) were observed in its gHMBC spectrum confirming that galactosylation occurred exclusively on the C4-OH of diol 6. The anomeric configurations of newly formed glycosyl linkages were established by coupling constants of anomeric protons (Gal 4.99 ppm, ³J_H1,H2 = 7.8 Hz indicating β linkage; Fuc 4.64 ppm, ³J_H1,H2 = 4.2 Hz suggesting α linkage).

Recently, an elegant synthesis of Lewis^X pentasaccharide⁹ was reported using the automated solid phase synthesis method pioneered by Seeberger and coworkers.³² The glycoassembly process on the carbohydrate synthesizer took 18 hours using 10 – 15 eq. of each glycosyl building block with a 12.7% overall yield. As a comparison, through one pot synthesis, Lewis^X was assembled rapidly with higher yield without resorting to the usage of large excess of building blocks, which can be very tedious to prepare. Furthermore, the progress of the one pot synthesis can be easily followed by TLC allowing convenient reaction monitoring and the intermediate oligosaccharide can be readily characterized as demonstrated by trisaccharide 10. This highlights that our one-pot method complements well with the existing automated solid phase synthesis method.

For a highly convergent synthesis of dimeric Lewis^X, we designed three modules, trisaccharide donor 10, bifunctional trisaccharide acceptor 13 and lactoside 7. To simplify the overall synthetic design without relying on selective activation of different types of glycosyl donors,⁷ the same aglycon leaving group p-STol was used for 10 and 13. If the traditional armed-disarmed chemoselective glycosylation approach^31,33 were to be applied, the acceptor must be less reactive than the glycosyl donor. This would require manipulations of the protective groups to significantly decrease the relative anomeric reactivity of the trisaccharide acceptor 13, which can be difficult due to the large number of protective groups present. Because donor activation and addition of acceptor occur at two distinct steps using the pre-activation based method, anomeric reactivities of glycosyl donor and acceptor are independent of each other.¹³ This would allow the direct glycosylation of trisaccharide 13 by trisaccharide 10, even though 10 is less reactive.

Diol 13 was prepared by hydrazine acetate treatment of trisaccharide 12, which was obtained by one pot sequential reactions of galactoside 4 with glucosamine 5 and fucose 6 in a similar fashion as the formation of 10 (Table 1, entry 3). With all the necessary building blocks in hand, synthesis of dimeric Lewis^X octasaccharide was carried out in one pot (Table 1, entry 4). The trisaccharide donor 10 was preactivated by p-TolSCl/AgOTf, followed by addition of acceptor 13. After complete consumption of 13 was confirmed by TLC analysis, the lactose acceptor 7 was added followed by another equivalent of p-TolSCl/AgOTf. The desired fully protected dimeric Lewis^X octasaccharide 14 was successfully acquired in 44 – 61% yield. Glycosylation on 3-OH of trisaccharide 13 was confirmed by ¹H-NMR analysis of the p-nitrobenzoate derivative of 14 (octasaccharide 15) with its H_2d proton appearing at 5.25 ppm as a triplet (³J = 9.0 Hz).

Deprotection of Lewis^X pentasaccharide 11 was performed by removal of benzoyl and Phth with ethylenediamine, followed by selective acetylation of the free amine leading to pentasaccharide 16 (Scheme 1a). Attempts to simultaneously reduce the azido moiety and benzyl groups in 16 through catalytic hydrogenation with Pd/C or Pd(OH)₂ under atmospheric pressure or high pressure (100 ~ 250 psi) hydrogen gas failed to yield any desired product. ¹H-NMR of the reaction mixture showed complex mix of compounds. Instead, Staudinger reduction of the azide moiety in 16 by trimethylphosphine under basic condition gave the amine,¹⁷ which underwent smooth hydrogenolysis with Pd(OH)₂ to produce desired fully deprotected Lewis^X 1 as an acetate salt.

Scheme 1.

Deprotection of Lewis^X and Dimeric Lewis^X

For dimeric Lewis^X deprotection, removal of benzoyl and Phth from 14 and selective acetylation were performed under same conditions as those for Lewis^X 11. Subsequent Staudinger reduction of 17 under basic condition, however, led to partial removal of one acetamido moiety. As an alternative, compound 17 was treated with trimethylphosphine in aqueous THF without any base to give a free amine, which was hydrogenated to produce dimeric Lewis^X octasaccharide 2 in 35% overall yield for the four steps (Scheme 1b).

In conclusion, we have demonstrated that branched oligosaccharides can be constructed using the combination of pre-activation and reactivity based one pot synthesis with exclusive regio- and stereo-selectivities. High synthetic efficiency was achieved without requiring large excess of building blocks. Single type of glycosyl donors, i.e., p-tolyl thioglycosides was used for all glycosylations, thus significantly simplifying overall synthetic design. With its anomeric reactivity independent nature, the pre-activation based chemoselective glycosylation method presents a powerful strategy for modular synthesis of complex oligosaccharides.

Experimental Section

3-Azidopropyl (2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranosyl)-(1→4)-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)]-(6-O-benzyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (11)

Galactose 3 (100 mg, 0.172 mmol) was dissolved in DCM (3 mL) and stirred at −78 °C with freshly activated molecular sieves MS 4Å (100 mg) for 30 minutes. Silver triflate (173 mg, 0.67 mmol) dissolved in acetonitrile (0.3 mL) was added to the reaction mixture. Five minutes later, orange-colored p-TolSCl (27 μL, 0.172 mmol) was added directly into the reaction mixture. This needs to be performed quite quickly in order to prevent the p-TolSCl from freezing inside the syringe tip or on the flask wall. The yellow color of the solution quickly dissipated within one minute, indicating the complete consumption of p-TolSCl and the complete activation of galactose donor 3 was confirmed by TLC analysis. The glucosamine acceptor 4 (78 mg, 0.155 mmol) along with TTBP (43 mg, 0.172 mmol) dissolved in DCM (2 mL) was then added drop wise to the reaction mixture. This was stirred for 20 minutes at which point the glucosamine acceptor 4 was completely consumed. The fucose donor 5 (93 mg, 0.172 mmol) and TTBP (43 mg, 0.172 mmol) dissolved in Et₂O (2 mL) were added to the mixture. After 10 minutes, p-TolSCl (27 μL, 0.172 mmol) was added into the reaction mixture which was stirred for an additional 45 minutes. When complete consumption of the fucose donor 5 was confirmed by TLC analysis, the lactose acceptor 6 (116 mg, 0.120 mmol) and TTBP (43 mg, 0.172 mmol) were dissolved in DCM (2 mL) and added to the reaction mixture. This was stirred for 10 minutes at −78 °C and then silver triflate (44 mg, 0.172 mmol) was added. After five minutes, p-TolSCl (27 μL, 0.172 mmol) was added into the reaction mixture which was stirred for 45 minutes. The mixture was then filtered through Celite and the Celite was washed with DCM until no organic compounds were present in the filtrate. The filtrate was extracted with saturated solution of NaHCO₃. The organic layer was then dried over Na₂SO₄ and concentrated to dryness. The residue was purified by silica gel column chromatography (Hexanes/DCM/EtOAc, 5:3:2). The desired product was obtained in 40–60% yield as a white solid. ${[\alpha ]}_{\text{D}}^{25}$-44 (c 1.5, CH₂Cl₂) ¹H-NMR (600 MHz, CDCl₃): δ 8.04-6.76 (m, 69H), 5.85 (t, 1H, ³J= 9.0 Hz), 5.56 (s, 1H), 5.19 (d, 1H, ³J = 7.8 Hz), 5.17 (dd, 1H, ³J = 4.8, 13.8 Hz), 5.12 (d, 1H, ³J = 12 Hz), 5.02 (d, 1H, ³J = 8.4 Hz), 4.88 (m, 2H), 4.81 (t, 2H, ³J = 10.2 Hz), 4.75 (d, 1H, ³J = 10.8Hz), 4.61 (d, 1H, ³J = 4.8 Hz), 4.54-4.49 (m, 4H), 4.44-4.29 (m, 6H), 4.27 (d, 1H, ³J = 11.4 Hz), 4.19-4.06 (m, 7H), 4.03-3.99 (m, 2H), 3.96 (s, 1H), 3.89 (dd, 1H. ³J = 2.4, 10.2 Hz), 3.84-3.80 (m, 2H), 3.79 (s, 1H), 3.76 (d, 1H, ³J = 4.8 Hz), 3.62 (d, 1H, ³J = 10.2 Hz), 3.55 (dd, 1H, ³J = 4.8, 10.2 Hz), 3.49-3.43 (m, 2H), 3.41-3.37 (m, 5H), 3.32-3.29 (m, 4H), 3.28 (q, 1H, ³J = 7.8, 9.0 Hz), 3.20 (d, 1H, ³J = 10.8 Hz), 3.16 (s, 1H), 2.86 (d, 1H, ³J = 8.4 Hz), 1.80-1.77 (m, 2H), 1.26 (d, 3H, ³J = 6.6 Hz); ¹³C-NMR (150 MHz, CDCl₃) δ 166.3, 164.9, 139.7, 139.7, 139.6, 139.2, 138.8, 138.6, 138.6, 138.5, 138.4, 138.2, 137.8, 130.1, 129.9, 129.5, 129.3, 128.9, 128.8, 128.7, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.1, 128.0, 127.9, 127.9, 127.9, 127.9, 127.8, 127.8, 127.7, 127.6, 127.4, 127.3, 127.2, 127.1, 127.0, 126.9, 126.4, 125.9, 103.6, 102.8 (¹J_C-H = 163.4 Hz), 100.0 (¹J_C-H = 161.3 Hz), 99.9 (¹J_C-H = 158.2 Hz), 99.8 (¹J_C-H = 164.6 Hz), 97.6 (¹J_C-H = 174.1 Hz), 83.1, 82.2, 81.8, 79.2, 78.7, 77.5, 77.2, 77.0, 76.8, 76.2, 75.3, 75.2, 75.0, 74.9, 74.8, 74.1, 73.6, 73.5, 73.2, 73.0, 72.9, 71.6, 66.7, 66.6, 66.5, 48.5, 29.4, 16.5; HRMS [M+Na]⁺ m/z: calcd for C₁₃₂H₁₃₂N₄NaO₂₈ 2244.8926, found 2244.8960.

3-Aminopropyl β-D-galactopyranosyl-(1→4)-[(α-L-fucopyranosyl)-(1→3)]-(2-N-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranoside (1)

The fully protected pentasaccharide 11 (200 mg, 0.09 mmol) was mixed with ethylenediamine (0.5 mL) in nbutanol (5 mL). The reaction was stirred at 130 °C for 20 hours. The mixture was then concentrated and the residue was dissolved in DCM and extracted with a saturated solution of NH₄Cl. The organic layer was dried over Na₂SO₄ and concentrated to dryness. The crude residue was purified by silica gel column chromatography. The desired product was obtained in its pure form as an off-white solid. The newly formed compound was then dissolved in MeOH (5mL) along with triethylamine (0.1 mL) and acetic anhydride (0.1 mL, 15 eq). The reaction mixture was stirred at room temperature for 4 hours. It was then concentrated and the residue was dissolved in DCM and extracted with a saturated solution of NH₄Cl. The organic layer was dried over Na₂SO₄ and concentrated to dryness. The crude residue was purified by silica gel column chromatography (Hexanes/EtOAc, 1:1→1:3) to afford the pentasaccharide 16 as a white solid. A mixture of 16 (100 mg, 0.052 mmol), 1 M solution of PMe₃ in THF (0.360 mL, 7 eq) and 0.1 M NaOH (0.5 mL) in THF was stirred at 60 °C overnight. The mixture was then concentrated and the resulting residue was dissolved in DCM and extracted twice with H₂O. The organic layer was dried over Na₂SO₄ and concentrated to dryness. The crude product was purified by silica gel column chromatography (DCM → DCM/MeOH 8:1). The desired product bearing a terminal free amine was obtained as an off-white solid. Finally a mixture of this product (80 mg, 0.042 mmol) and Pd(OH)₂ (80 mg) in DCM/MeOH/H₂O/AcOH (1 mL: 1 mL: 2 mL: 2 mL) was stirred at room temperature under atmospheric pressure H₂ for 24 hours. The mixture was filtered through Celite, concentrated and extracted with DCM (3 times) and EtOAc (3 times). The water layer was then lyophilized to afford a white solid which was purified by Sephadex G-10 size exclusion column. The pure Lewis^X pentasaccharide 1 was obtained in acetate form as a solid in 53% yield over four steps. ${[\alpha ]}_{\text{D}}^{25}$-217 (c 0.5, H₂O) ¹H-NMR (600 MHz, D₂O) δ 4.94 (d, 1H, ³J = 3.6 Hz), 4.52 (d, 1H, ³J = 8.4 Hz), 4.33 (d, 1H, ³J = 8.4 Hz), 4.28 (d, 1H, ³J= 8.4 Hz), 4.25 (d, 1H, ³J = 8.4 Hz), 3.97 (d, 1H, ³J = 2.4 Hz), 3.80-3.75 (m, 4H), 3.73-3.69 (m, 4H), 3.63-3.37 (m, 20H), 3.31 (t, 1H, ³J = 7.2 Hz), 3.16-3.13 (m, 2H), 2.97 (t, 2H, ³J= 7.2 Hz), 1.83 (s, 3H), 0.99 (d, 3H, ³J = 6.0 Hz); ¹³C-NMR (150 MHz, D₂O) δ 181.7, 174.8, 103.0, 102.7, 102.2, 101.9, 98.7, 82.2, 78.4, 75.2, 75.1, 75.0, 74.9, 74.4, 73.1, 72.8, 72.6, 72.0, 71.1, 70.0, 69.3, 68.5, 68.4, 68.0, 67.8, 66.8, 61.6, 61.1, 60.1, 59.7, 56.1, 37.7, 26.8, 23.4, 22.4, 15.4; HRMS [M+Na]⁺ m/z: calcd for C₃₅H₆₂N₂NaO₂₅ 933.3539, found 933.3531.

Supplementary Material

1File002. Supporting information Available.

Experimental procedures. Selected ¹H-, ¹³C- and 2D NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.