Total Synthesis of the Aminopropyl Functionalized Ganglioside GM₁

Abstract

GM1 is a common ganglioside pentasaccharide present on mammalian cell surface. It has been shown to play important roles in cellular communications and initiation of β-amyloid aggregation. In order to synthesize GM1, an efficient synthetic route was developed via a [3+2] strategy. The GM3 trisaccharide acceptor bearing an azido propyl group at the reducing end was prepared using the traditional acetamide protected sialyl thioglycosyl donor, which gave better stereoselectivity than sialyl donors protected with trichloroacetamide or oxazolidinone. The glycosylation of the axial 4-hydroxyl group of GM3 by the disaccharide donor was found to be highly dependent on donor protective groups. Donor bearing the more rigid benzylidene group gave low glycosylation yield. Replacing the benzylidene with acetates led to productive coupling and formation of the fully protected GM1 pentasaccharide. Deprotection of the pentasaccharide produced GM1 functionalized with the amino propyl side chain, which will be a valuable probe for biological studies.

1 Introduction

GM₁ (1a), a member of the ganglioside family, is commonly present in vertebrate plasma membranes and especially enriched in nerve tissues[1, 2]. There are many significant biological functions ascribed to GM₁, such as receptor for pathogen binding[3], cell-growth modulators[4], and neurotrophic factor[5]. Furthermore, GM₁ has been proposed to be a nucleating site for initiating β-amyloid aggregation, which is implicated in the development of Alzheimer’s diseases (AD) [6–10]. GM₁ level showed significant increase in amyloid positive nerve terminals obtained from the cortex of AD patients[11]. In order to better understand the roles of GM₁ in β-amyloid aggregation and AD development, sufficient quantity of GM₁ is needed. Herein, we report our synthesis of an aminopropyl side chain functionalized GM₁ derivative (1b), which can be readily used for bioconjugation.

2 Experimental

2.1 General experimental section

General Experimental Procedures

All reactions were carried out under nitrogen with anhydrous solvents in flame-dried glassware, unless otherwise noted. All glycosylation reactions were performed in the presence of molecular sieves, which were flame-dried right before the reaction under high vacuum. Glycosylation solvents were dried using a solvent purification system and used directly without further drying. Chemicals used were reagent grade as supplied except where noted. Analytical thin-layer chromatography was performed using silica gel 60 F254 glass plates; spots were visualized by UV light (254 nm) and by staining with a yellow solution containing Ce(NH₄)₂(NO₃)₆ (0.5 g) and (NH₄)₆Mo₇O₂₄ 4H₂O (24.0 g) in 6% H₂SO₄ (500 mL). Flash column chromatography was performed on silica gel 60 (230–400 Mesh). NMR spectra were referenced using Me₄Si (0 ppm), residual CHCl₃ (¹H-NMR 7.26 ppm, ¹³C-NMR 77.0 ppm). Peak and coupling constant assignments are based on ¹H-NMR and ¹H–¹H gCOSY. High-resolution mass spectra were recorded on a Q-TOF Ultima API LC-MS instrument with Waters 2795 Separation Module (Waters Corporation, Milford, MA).

2.2 Synthesis of fully protected GM₁ pentasaccharide 2

The mixture of donor 3 (216 mg, 0.2 mmol), acceptor 4 (809 mg, 0.6 mmol) and freshly activated MS 4Å (600 mg) in dry CH₂Cl₂ (6 mL) was stirred for 30 minutes at room temperature, and cooled down to −70°C followed by the addition of AgOTf (154 mg, 0.6 mmol) in anhydrous acetonitrile (0.1 mL). After 5 minutes, p-TolSCl (29 μL, 0.2 mmol) was added via a microsyringe directly to the solution without touching the wall of reaction flask. The orange color of p-TolSCl dissipated within a minute. The reaction mixture was stirred for 1.5 h until the temperature reached −20°C and triethylamine (30 μL) was added. The mixture was diluted with CH₂Cl₂ (50 mL) and filtered through Celite. The filtrate was concentrated and purified by flash column chromatography (hexanes : EtOAc = 3 : 2) to give fully protected GM₁ 2 (267.6 mg, 58%). ¹H NMR (500 MHz, CDCl₃) δ 7.95 (s, 2 H), 7.54-6.99 (m, 45 H), 5.52-5.46 (t, 1 H), 5.44 (d, J = 7.0 Hz, 1 H), 5.39 (d, J = 3.0 Hz, 1 H), 5.25-5.20 (m, 2 H), 5.16 (d, J = 10 Hz, 2 H), 5.05-5.00 (m, 1 H), 4.94 ( d, J = 10 Hz, 2 H), 4.85 -4.76 (m, 3 H), 4.75-4.65 (m, 3 H), 4.63-4.52 (m, 4 H), 4.51-4.37 (m, 5 H), 4.36-4.30 (m, 2 H), 4.26-4.08 (m, 2 H), 4.05-3.76 (m, 11 H), 3.72-3.55 (m, 8 H), 3.54-3.47 (t, 2 H), 3.45-3.39 (m, 2 H), 3.38-3.36 (m, 5 H), 2.11-1.97 (m, 8 H), 1.96(s, 6H), 1.89 (s, 3 H), 1.88 (s, 3 H), 1.87-1.70 (m, 5 H); ¹³C NMR (125 MHz, CDCl₃) δ 170.9, 170.5, 170.47, 170.2, 168.7, 165.3, 162.2, 139.11, 139.1, 139.0, 138.9, 138.8, 138.5, 138.0, 133.2, 130.5, 130.1, 129.3, 128.6, 128.57, 128.5, 128.49, 128.4, 128.36, 128.2, 128.15, 127.8, 127.77, 127.7, 127.6, 127.5, 126.6, 126.5, 103.7, 102.7, 100.9, 100.6, 100.5, 83.1, 82.5, 81.9, 80.3, 76.8, 76.0, 75.6, 75.5, 75.45, 75.4, 74.0, 73.4, 73.3, 73.1, 73.0, 72.9, 72.3, 71.8, 70.2, 69.4, 69.1, 68.5, 67.7, 67.6, 67.2, 66.7, 66.4, 62.4, 53.1, 51.8, 48.6, 29.9, 25.5, 21.7, 21.0, 20.9, 20.8. ESI-MS: [M + Na]⁺ C₁₁₇H₁₃₂Cl₃N₅NaO₃₇ calcd 2326.8, obsd 2327.0.

2.3 Deprotected GM₁ (1b)

The mixture of 2 (231 mg, 0.1 mmol), 1 M NaOH (2 mL, 2 mmol), and THF (15 mL) was stirred at 50 °C overnight and then concentrated to dryness. The resulting residue was diluted with CH₂Cl₂ (100 mL), and the organic phase was washed by H₂O and then dried over Na₂SO₄, filtered, and concentrated to dryness. The resulting residue was dissolved in a mixture of methanol (20 mL) and trie-thylamine (0.14 mL, 1 mmol). Acetic anhydride (0.094 mL, 1 mmol) was added dropwise, and the mixture was stirred at room temperature for 4 h. The reaction was quenched by adding a few drops of H₂O and then diluted with EtOAc (100 mL). The organic phase was washed with a saturated aqueous solution of NaHCO₃ and H₂O, dried over Na₂SO₄, filtered, and concentrated to dryness. Silica gel column chromatography (hexanes : EtOAc = 2:1) afforded the N-acetylated product as a white solid. The mixture of the N-acetylated product, 1 M of PMe₃ in THF (0.2 mL, 0.056 mmol), 0.1 M NaOH (2 mL, 0.5 mmol), and THF (12 mL) was stirred at 60 °C under N₂ overnight. The mixture was concentrated, and the resulting residue was diluted with CH₂Cl₂ (150 mL). The organic phase was washed with H₂O and then dried over Na₂SO₄, filtered, and concentrated to dryness. The resulting residue was purified by quickly passing through a short silica gel column (CH₂Cl₂ : MeOH = 10:1). The mixture of the obtained solid and Pd(OH)₂ in MeOH/H₂O (10 mL:3 mL) was stirred under H₂ at room temperature overnight and then filtered. The filtrate was concentrated to dryness under vacuum. The aqueous phase was further washed with CH₂Cl₂ (5 mL × 3) and EtOAc (5 mL × 3), and the aqueous phase was dried under vacuum to afford 1b (acetate salt) as a white solid (68.6 mg, 65% for four steps). ¹H NMR (500 MHz, D₂O) δ 4.71 (d, 1 H, J = 8.5 Hz), 4.50-4.43 (m, 3 H), 4.12-4.05 (m, 3 H), 4.01-3.89 (m, 3 H), 3.85 (d, 1 H, J = 3.5 Hz), 3.81 (dd, 1 H, J = 13.0, 4.0 Hz), 3.79-3.65 (m, 14 H), 3.65-3.50 (m, 7 H), 3.46-3.41 (m, 2 H), 3.32-3.23 (m, 2 H), 3.07 (t, 2 H, J = 6.6 Hz), 2.60 (dd, 1 H, J = 12.5, 4.0 Hz), 1.97 (s, 3 H), 1.94 (s, 3 H), 1.94 (t, 1 H, J = 12 Hz), 1.90-1.84 (m, 2 H). ¹³C NMR (125 MHz, D₂O) δ 181.6, 175.1, 174.9, 174.2, 104.8, 102.7, 102.6, 102.2, 101.8, 80.5, 78.7, 77.2, 75.0, 74.9, 74.5, 74.2, 73.2, 72.8, 72.6, 72.4, 70.8, 68.8, 68.7, 68.1, 68.0, 63.0, 61.2, 61.1, 60.8, 60.2, 51.7, 51.3, 37.7, 27.1, 23.4, 22.7, 22.2. HRMS: [M+Na]⁺ C₄₀H₆₉N₃NaO₂₉ calcd 1078.3914, obsd 1078.3942.

3 Results and discussion

Although synthesis of ganglioside GM₁ and its derivatives had previously been accomplished by several laboratories[12–17], it is still a challenging task. The main difficulty lies in the construction of the bifurcating branches onto the reducing end lactoside. As the sialic acid and the galactose (Gal)-galactosamine (GalN) disaccharide are linked to neighboring hydroxyl groups, the installation of one branch can pose serious steric hindrance for introduction of the other. We decided to attach the sialic acid first as sialyl donors are known to have lower anomeric reactivities than common pyranosyl donors due to the presence of the electron withdrawing carbonyl group at the anomeric center[18, 19] (Figure 1). Based on this consideration, the key step of our synthesis will be the coupling of the Gal^E-GalN^D disaccharide fragment 3 with a selectively protected sialyllactose block 4 (GM₃ chain).

Figure 1.

Retrosynthetic Analysis for Synthesis of GM₁ 1b.

To synthesize the GM₃ trisaccharide, the lactosyl diol 8 bearing multiple benzyl ethers as the protective groups was selected as the acceptor, which should have good nucleophilicity due to the electron donating nature of the multiple benzyl ether groups present. The synthesis of lactose acceptor 8 began with the commercially available lactose 9 (Scheme 1). Acetylation of 9 in the presence of sodium acetate and acetic anhydride gave the per-acetylated lactose 10, which underwent BF₃·Et₂O promoted glycosylation with 3-chloropropan-1-ol to provide the desired β-lactoside 11[20]. SN₂ displacement of the chloride with sodium azide followed by Zemplen deacetylation and regioselective isopropylidene formation produced lactoside 14. Global protection of the free hydroxyl groups as benzyl ethers and acid mediated isopropylidene removal led to the desired lactosyl diol acceptor 8[21] in 85% yield for the two steps.

Scheme 1.

Synthesis of the lactosyl acceptor 8.

Sialylation of the lactosyl disaccharide 8 was tested next. Besides the aforementioned low reactivity of sialyl donor, another difficulty in sialylation is stereochemical control as the naturally existing sialyl linkage is the thermodynamically less favored α linkage[19]. Recently, it was discovered that the installation of an electron withdrawing protective group including TCA and oxazolidinone on the 5-N position significantly enhanced the reaction yield and stereoselectivity[22, 23]. Thus, we investigated three sialyl donors 16[24], 17[24] and 18[25] with their 5-N-acetyl group substituted with more electron withdrawing protective groups. As shown in scheme 2, donor 16 gave excellent stereoselectivity, albeit with a low yield (Scheme 2a). Changing the anomeric leaving group from STol in 16 to trifluoroacetimidate (donor 17) did not improve the situation much (Scheme 2b). On the other hand, the usage of the oxazolidinone protected sialyl donor 18 gave 76% yield of the trisaccharide as a 2:1 mixture of α:β anomers, which were difficult to separate (Scheme 2c). Instead of further optimizing the glycosylation reactions using these donors, we examined the 5-N-acetamide donor 7[24], as it was simpler to prepare than donors 16–18. Interestingly, donor 7 gave 60% yield and exclusive α selectivity in coupling with the lactose acceptor 8 (Scheme 2d).

Scheme 2.

Synthesis of GM₃ trisaccharide acceptor.

After establishing a viable route to GM₃, we assessed the formation of the second branch. The Gal-GalN disaccharide building block 3 was accessed by the reaction of galactosyl donor 5[26] and galactosaminyl acceptor 6[27]. To prepare compound 6, the amino group of GalN 21 was protected by the trichloroethoxocarbonyl (Troc) group followed by peracetylation to give 22 (Scheme 3a). The anomeric acetate in 22 was replaced with p-toluenethiol as promoted by BF₃·Et₂O to yield compound thioglycoside 23. Zemplen reaction using sodium methoxide removed all the acetyl groups in 23 followed by benzylidenation of the newly liberated 4,6-hydroxyl groups led to the galactosylaminyl acceptor 6. It was important to maintain low temperature for this reaction to avoid the possible side reaction of Troc with sodium methoxide. As both 5 and 6 are thioglycosides, the glycosylation of 6 by 5 was performed under the pre-activation condition[28] to avoid the activation of the acceptor or the product. Treatment of donor 5 with AgOTf/p-TolSCl[27] at −78 °C in the presence of a sterically hindered base tri-^tbutylpyrimidine (TTBP)[27] cleanly activated the donor within a few minutes. Addition of acceptor 6 to the activated donor solution led to the formation of disaccharide 24 in 66% yield (Scheme 3b).

Scheme 3.

Synthesis of a) the galactosaminyl acceptor 6; and b) Gal-GalN disaccharides 24 and 3.

The union of the Gal-GalN disaccharide 24 and GM₃ trisaccharide 4 was then explored, which failed to produce the desired GM₁ pentasaccharide. We hypothesized this was because upon donor activation, the Troc moiety in donor 24 participates in stabilizing the oxacarbenium ion through the formation of a five membered oxazoline ring (Scheme 4a). In order to accommodate this, the pyranosyl ring of GalN needs to undergo conformational changes. However, the presence of the benzylidene ring on the GalN conformationally rigidifies the ring[29], thus hindering conformational changes in the pyranosyl ring and lowering the reactivity of the activated donor towards the sterically hindered GM₃ acceptor. To overcome this difficulty, the benzylidene group was removed from disaccharide 24 and the free hydroxyl groups were acetylated (disaccharide 3). Gratifyingly, glycosylation of donor 3 with GM₃ trisaccharide 4 proceeded to give the fully protected GM₁ pentasaccharide 2 in 58% yield (70% based on acceptor consumed) (Scheme 4b), with the correct molecular weight of 2327.0 [M + Na]⁺ given by mass spectrometry. NMR spectra of compound 2 showed broad peaks, which was presumably due to the presence of multiple conformations at room temperature.

Scheme 4.

a) Formation of the 5-membered oxazoline ring by activated donor 24 was presumably difficult due to the presence of the rigid benzylidene ring, thus lowering the reactivity of activated 24 towards acceptor 4. b) Formation of the fully protected GM₁ pentasaccharide 2. c) Deprotection of pentasaccharide 2.

The deprotection of pentasaccharide 2 began with the removal of the Troc, acyl and ester protecting groups using 1 M NaOH in THF overnight (Scheme 4c). The newly liberated amine was selectively acetylated in the presence of multiple hydroxyl groups by acetic anhydride in methanol. Staudinger reduction of the azide group and subsequent catalytic hydrogenation over Pearlman’s catalyst gave the fully deprotected GM₁ analog 1b in 65% overall yield for all deprotection steps. The α linkage between sialic acid and the lactose unit was confirmed by the NMR coupling constant between C₁ and H_3ax of sialic acid (³J(C₁,H_3ax) = 5.7 Hz)[27]. The β linkages for the rest of the glycosidic bonds were supported by the one bond coupling constants between the respective anomeric carbon and proton (163.5 Hz, 162 Hz, 162 Hz, 164 Hz)[27]. Correlations of the anomeric carbon of the GalN^D unit (102.6 ppm) with H₄ of Gal^B (4.06 ppm) and the anomeric carbon of the sialic acid unit (101.8 ppm) with H₃ of Gal^B (4.08 ppm) were found in HMBC NMR spectrum, thus confirming the regiochemistry of 1b.

4 Conclusions

In conclusion, a stereo- and regio-controlled total synthesis of aminopropyl functionalized GM₁ was achieved. Compared to previous synthesis, our method only employed a single type of glycosyl donors, i.e., thioglycosides, which simplified overall building block design. With the aminopropyl side chain, our GM₁ analog can be readily conjugated to liposomes and nanoparticles. This will be very useful for deciphering the role of GM₁ ganglioside in the induction of β-amyloid aggregation as well as pathogen infection. The results from those studies will be reported in due course.