Christian Gege, Momo Arsic, Richard R. Schmidt and Armin Geyer

Received: 7 August 2000 / Uploaded: 10 August

Glycolipids cover the surface of living cells and determine their properties in cell-adhesion processes. The extremely complex and heterogeneous native cell membrane forbids the functional characterization of individual glycolipid components. Homogeneous neoglycolipids on model membranes reduce the complex native glycocalix to its essential features.

Figure 1. 1,2-di-O-alkyl-sn-glycerol serves as the membrane anchor for lactose building blocks. The triethyleneglycol spacer mimics one lactose disaccharide. Although the number of bonds are the same (red), the flexibility of the triethyleneglycol spacer is much higher.

Fig. 2 gives an outline of the synthetic route to the 1,2-di-O-hexadecyl-sn-glycerol derivatives Peg_n (n = 3, 6, and 9).³ Benzyl protected triethylene glycol derivative 1 was synthesized in one step from commercially available triethylene glycol monochlorohydrin. The spacered lipid Peg₃ was synthesized in high overall yield by the coupling of 1 (2.5 equivalents) and 1,2-O-isopropylidene-sn-glycerol in the presence of sodium hydride as base and tetrabutylammonium iodide (TBAI) as activator, followed by acid catalyzed de-O-isopropylidenation, di-O-alkylation with hexadecyl bromide under the above described conditions, and finally hydrogenolytic O-debenzylation. Repetitive coupling of Peg₃ with the building block 1 (2.5 equivalents) under the above described conditions yielded, after de-O-benzylation, the elongated lipids Peg₆ and Peg₉.

Figure 2. Synthesis of ethylene glycol spacered 1,2-Di-O-hexadecyl-sn-glycerol derivatives Peg_n (n = 3,6, and 9).

The synthetic route to these glycolipids is shown in Fig. 3. Glycosylation of known lactose trichloroacetimidate 2¹ with 1,2-di-O-hexadecyl-sn-glycerol^2,3 as lipid anchor was performed in the presence of BF₃�Et₂O as catalyst to afford the lactose intermediate 3 in 94 % yield. The b-configuration of the newly formed glycosidic bond was confirmed in the ¹H NMR spectrum (J_1,2 = 7.9 Hz). Acid-catalyzed cleavage of the isopropylidene group furnished 4, which served as acceptor for the next glycosylation. Removal of the O-benzoyl protective groups with sodium methoxide in CH₂Cl₂/CH₃OH afforded Lac₁ in almost quantitative yield after purification on reversed phase (RP-18) silica gel.

Elongation of 4 with donor 2 in the presence of BF₃�Et₂O as catalyst at � 20�C afforded exclusively the expected b(1®3)-connected bis-lactose intermediate 5 (J_1,2 = 7.9 Hz for 1c-H; shift of the 4b-H-signal from 3.85 ppm to 5.29 ppm after acetylation). Again, de-O-isopropylidenation furnished the following acceptor 6, and de-O-benzoylation the desired Lac₂ derivative under the conditions described above. Repetitive coupling of 6 with donor 2 yielded the elongated protected glycolipid 7, which was de-O-isopropylidenated in the same manner. Due to the low solubility of Lac₃ the de-O-benzoylation and purification was performed in dimethyl sulfoxide (DMSO) mixtures.

Figure 3. Synthesis of Lac_n (n = 1, 2, and 3) by repetitive glycosylation.

Fig. 4 shows mixed neoglycolipids characterized by triethylene spacers of various lengths inserted between lactose and the membran anchor. They were named LacPeg_n, with n corresponds to the number of ethylene glycol units (n = 3, 6, or 9). The benzoylated lactose trichloroacetimidate 9 is available from lactose in a three-step procedure.⁴ Again, the benzoyl protecting group next to the anomeric activation directs the formation of b-linked glycolipids without formation of undesired orthoester.

Glycosidation of 1,2-di-O-hexadecyl-sn-glycerol derivatives of Peg_n (n = 3, 6, 9) as acceptors was performed with donor lactose trichloroacetamidate 9 in the presence of TMSOTf as catalyst to afford the protected products 10, 11, and 12, respectively. The newly formed glycosidic bond was found exclusively b -oriented in the ¹H-NMR spectrum. Removal of the benzoyl groups with sodium methoxide in CHCl₃/CH₃OH affored the LacPeg_n derivatives in quantitative yields after purification by flash chromatography.

Figure 4. Synthesis of the spacered lactosyl-glycolipids.

Glycosylation of Peg₉ with the glucosamine imidate⁵ led to b-linked glycoside (J_1,2 = 8.6 Hz) in high yield. Treatment with activated zinc in deuterated acetic anhydride led to the N-acetyl derivative, which furnished on treatment with NaOMe in MeOH the desired glucosamine derivative GlcNAcPeg₉.³

Figure 5: Synthesis of GlcNAcPeg₉.

So-called bicelles are a powerfull tool for the spectroscopic characterization of neoglycolipids. This membrane model was developed by Prestegard for NMR studies.⁶ Bicelles combine several advantageous qualities. They form a flat and highly solvated bilayer model which is stable between 303 and 320 K and which can be studied with highfield NMR methods (Fig. 6). Fig. 7 shows typical phase transitions when glycolipid GlcNAcPeg₉ is inserted into the membranes. In the temperature range of bicelle formation, two ³¹P NMR signals are observed which belong to the edge (lowfield, mainly dihexanoylphosphatidylcholine, DHPC) and the flat bilayer (highfield, mainly dimyristoylphosphatidylcholine, DMPC). The ¹H NMR shows excessive line-broadening. Also the carbohydrate protons resonances are broadened, proving the slowing down of the glycolipid tumbling. The temperture of phase transition is influenced by the admixed glycolipid. ³¹P NMR spectra and ¹H NMR spectra are shown in Fig. 7 for GlcNAcPeg₉. Only in a very small region of less than 5 K the bicelles are formed. The ³¹P NMR spectra exhibit two resonances: the broader highfield signal originates from the flat bilayer, while the sharper lowfiled signal comes from the DHPC at the edges of the bicelles. Only one isotropic ³¹P resonance is detected above and below this temperature range. At 302 K all three signals are visible and indicate the existence of bicelles plus isotropic liposomes. A very sharp phase transition is observed between 305 K and 306 K, the bicelles `melt�.

Figure 6: Disc-shaped micelles, so-called bicelles are formed by DMPC and DHPC. Flat lipid bilayers are formed by the DMPC fraction, while the DHPC molecules probably concentrate at the edges of the bicelles. The bicelles form a nematic fluid-crystalline phase with the average disc vector perpendicular to the direction of the magnetic field B₀. The diameter of the disc varies from 10 to 100 nm, the thickness is about 4 nm.

Figure 7: Temperature dependence of a 3:1 mixture of DMPC and DHPC containing the neoglycolipid GlcNAcPeg₉ in a mM concentration.

The amphiphilic lactose-derived neoglycolipids described here exhibit poor solubilities in monolayered or bilayered membrane models. The triethylene spacer improves the solubilities. Although we described a straightforward synthetic strategy making the final compounds accessible in the gram scale, their NMR spectroscopic characterization in flat phospholipid bilayers remains a difficult task.

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