Total Synthesis of the Aminopropyl Functionalized Ganglioside GM1
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 \xce\xb2-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<!>General Experimental Procedures<!>2.2 Synthesis of fully protected GM1 pentasaccharide 2<!>2.3 Deprotected GM1 (1b)<!>3 Results and discussion<!>4 Conclusions
<p>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.</p><p>
</p><!><p>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).</p><!><p>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.</p><!><p>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.</p><!><p>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 GalE-GalND disaccharide fragment 3 with a selectively protected sialyllactose block 4 (GM3 chain).</p><p>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.</p><p>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).</p><p>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).</p><p>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.</p><p>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 H3ax of sialic acid (3J(C1,H3ax) = 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 GalND unit (102.6 ppm) with H4 of GalB (4.06 ppm) and the anomeric carbon of the sialic acid unit (101.8 ppm) with H3 of GalB (4.08 ppm) were found in HMBC NMR spectrum, thus confirming the regiochemistry of 1b.</p><!><p>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.</p>