An easy α-glycosylation methodology for the synthesis and stereochemistry of mycoplasma α-glycolipid antigens

Mycoplasma fermentans possesses unique α-glycolipid antigens (GGPL-I and GGPL-III) at the cytoplasm membrane, which carry a phosphocholine group at the sugar primary (6-OH) position. This paper describes a practical synthetic pathway to a GGPL-I homologue (C16:0) and its diastereomer, in which our one-pot α-glycosylation method was effectively applied. The synthetic GGPL-I isomers were characterized with 1H NMR spectroscopy to determine the equilibrium among the three conformers (gg, gt, tg) at the acyclic glycerol moiety. The natural GGPL-I isomer was found to prefer gt (54%) and gg (39%) conformers around the lipid tail, while adopting all of the three conformers with equal probability around the sugar position. This property was very close to what we have observed with respect to the conformation of phosphatidylcholine (DPPC), suggesting that the Mycoplasma glycolipids GGPLs may constitute the cytoplasm fluid membrane together with ubiquitous phospholipids, without inducing stereochemical stress.


Introduction
Mycoplasmas constitute a family of gram-positive microbes lacking rigid cell walls. They are suspected to be associated with human immune diseases, in either direct or indirect ways, although the molecular mechanism is not fully understood [1]. In recent biochemical studies, Mycoplasma outer-membrane lipoproteins [2,3] and glycolipids [4][5][6] are thought to serve not only as the main antigens but also as probable pathogens. Also in our research team, Matsuda et al. [7][8][9][10] isolated a new class of α-glycolipid antigens (GGPL-I and GGPL-III, Figure 1) from M. fermentans. Another α-glycolipid (MfGL-II), which has a chemical structure very close to GGPL-III, was identified and characterized by other groups [11][12][13][14]. Scheme 1: An established synthetic pathway to α-glycosyl-sn-glycerols 4a and 5a. A reagent combination of CBr 4 and Ph 3 P (Appel-Lee reagent) is utilized in either CH 2 Cl 2 or DMF as solvent. Absolute chemical structures of GGPL-I [15] and GGPL-III [16] have already been established by chemical syntheses of stereoisomers; these α-glycolipids have a common chemical backbone of 3-O-(α-D-glucopyranosyl)-sn-glycerol carrying phosphocholine at the sugar primary (6-OH) position. The fatty acids at the glycerol moiety are saturated, namely palmitic acid (C 16:0 ) and stearic acid (C 18:0 ). GGPL-I has a structural feature analogous to 1,2-di-O-palmitoyl phosphatidylcholine (DPPC) as a ubiquitous cell membrane phospholipid. Apparently, GGPLs are amphiphilic compounds that can form certain self-assembled structures under physiological conditions [12,13] and may give physicochemical stress on the immune system of the host [17]. In fact, our research team has proven that these α-glycolipid antigens have certain pathogenic functions [18,19].
In order to exploit their biological functions in detail, it is necessary to obtain these α-glycolipids in sufficient amounts. Thus, both genetic [20][21][22] and chemical synthetic approaches [23,24] are being followed, although no practical way has yet been established. In this paper, we report a chemical access to both a natural GGPL-I homologue (C 16:0 ) and its diastereomer I-a and I-b, in which our one-pot α-glycosylation methodology [25,26] is effectively applied. The two GGPL-I isomers prepared thereby were characterized with 1 H NMR spectroscopy, in terms of configuration and conformation at the asymmetric glycerol moiety.

Results and Discussion
A practical synthetic access to GGPL-I homologues GGPL-I provides two key asymmetric centers to be controlled, literally, in the synthetic pathway. One is the configuration at the chiral glycerol moiety, and another is the sugar α-glycoside linkage. In former synthetic works on 3-O-(α-D-glycopyranosyl)-sn-glycerol [27][28][29][30], chiral 1,2-O-isopropylidene-sn-glycerol has often been employed [29,30] as the acceptor substrate for different α-glycosylation reactions. In this case, however, attention should be paid to the acid-catalyzed migration of the dimethylketal group [23,[29][30][31]. In our synthetic pathway, chiral (S)-or (R)-glycidol is employed as an alternative source of the chiral glycerol to circumvent this problem. In an established synthetic approach, 6-O-acetyl-2,3,4-tri-O-benzyl protected sugar 1 [23] is used as the donor and treated with a reagent combination of CBr 4 and Ph 3 P (Appel-Lee reagent) in either CH 2 Cl 2 or DMF solvent, or a mixture of the two (Scheme 1). For the reaction in CH 2 Cl 2 , N,N,N',N'-tetramethylurea (TMU) is added after in situ formation of α-glycosyl bromide 2, which equilibrates with a more reactive β-glycosyl bromide species [32]. In the pathway using DMF, the α-glycosylation is routed via α-glycosyl cationic imidate 3, which was predicted in former studies [33] and evidenced in our preceding NMR and MS study [25,26].
A mixture of 4a and 5a was used in the following chemical transformation (Scheme 2). First, a lyso-glycolipid 6a was derived after deprotection at the sugar hydroxymethyl position and S N 2 substitution with cesium palmitate at the glycerol sn-1 position. Then, this compound was converted to glycolipid 8a after sequential reaction of the temporary tert-butyldimethylsilyl (TBDMS) -protected sugar, and O-acylation at the glycerol 2-OH position to give 7a, followed by removal of the TBDMS protecting group. For introducing the phosphocholine group at the sugar 6-OH position, we employed a phosphoroamidite method using 1H-tetrazole as a promoter [34]. First, 8a was treated with 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the presence of 1H-tetrazole, and then with choline tosylate to give 9a. After removal of the sugar O-benzyl group by catalytic hydrogenolysis, the GGPL-I homologue I-a was obtained. In the same way, the GGPL-I sn-isomer I-b was derived from a mixture of 4b and 5b available from the reaction between 1 and (R)-glycidol (Scheme 1 and Scheme 2b).

H NMR characterization of I-a, I-b and the related glycerolipids
1 H NMR spectroscopy provides a useful tool for discriminating between the two GGPL-I isomers as shown in Figure 2. A clear difference was observed in the chemical shifts of the glycerol methylene protons as designated by "a" and "b". Conversely, little difference was observed between the sn-isomers at the sugar H-1 signal as well as at the glycerol H-2 (Table 1).
Natural GGPL-I and GGPL-III gave 1 H NMR data very close to those of I-a, indicating that both have a common skeleton of 3-O-(α-D-glucopyranosyl)-sn-glycerol [15,16].
The glycerol moiety has two C-C single bonds. By free rotation, each of them is allowed to have three staggered conformers of gg (gauche-gauche), gt (gauche-trans) and tg (trans-gauche) ( Figure 3). In solution and also in self- contacting liquid-crystalline states, these conformers are thought to equilibrate with each other. In this study, we calculated time-averaged populations of the three conformers by means of 1 H NMR spectroscopy. As we reported in a preceding paper [41], the Karplus-type equation proposed by Haasnoot et al. [42] was adapted as follows: 2.8gg + 3.1gt + 10.7tg = 3 J H2,H1S (or 3 J H2,H3R ) 0.9gg + 10.7gt + 5.0tg = 3 J H2,H1R (or 3 J H2,H3S ) and gg + gt + tg = 1.

Conclusion
We have proposed a synthetic pathway to a GGPL-I homologue and its stereoisomer, in which our one-pot α-glycosylation methodology was effectively applied. We envisage that the simple method will allow us to prepare a variety of α-glycolipid antigens other than GGPLs and to prove their biological significance [43]. By the 1 H NMR conformational analysis, which was based on our former studies on deuteriumlabeled sn-glycerols and sugars, we have proven that GGPL-I and other 3-O-(α-D-glucopyranosyl)-sn-glycerolipids have a common conformational property at the chiral glycerol moiety: The lipid tail moiety prefers two gauche-conformations (gg and gt) in the order gt > gg >> tg, while the sugar head moiety adopts three conformers in an averaged population (gg = gt = tg). At the lipid tail position, the gt-conformer with clockwise helicity is predominant over the anticlockwise gg-conformer. The observed conformation was very close to what we have seen in DPPC ( Figure 5). Although these results were based on the solution state in a solvent mixture of CHCl 3 and CH 3 OH (10:1), it may be possible to assume that the mycoplasma GGPLs and the related 3-O-(α-D-glycopyranosyl)-sn-glycerolipids can constitute cytoplasm membranes in good cooperation with ubiquitous phospholipids without inducing stereochemical stress at the membrane.
The GGPL-I isomer I-b showed an overall conformational property similar to the natural isomer I-a and DPPC. However, it should be mentioned here that the chiral helicity of gt-conformers in I-b is reversed (anticlockwise) from the clockwise helicity of DPPC and GGPL-I. The difference in chirality seems critical in biological recognition events and also in physicochemical contact with other chiral constituents in cell membranes [44,45]. A typical procedure for the one-pot α-glycosylation: CBr 4 (1.6 g, 6.09 mmol) and Ph 3 P (2.02 g, 6.09 mmol) were added to a solution of 6-O-acetyl-2,3,4-tri-O-benzyl-D-glucose (1) (1.0 g, 2.03 mmol) in 10 mL of DMF and stirred for 3 h at rt. Then, (S)-glycidol (301 mg, 4.06 mmol) was added to the reaction mixture and stirred for 14 h at rt. Products were diluted with a mixture of toluene and ethyl acetate (10:1), and the solution was washed with saturated aq. NaHCO 3 and aq. NaCl solution, dried and concentrated. The residue was purified by silica gel column chromatography in toluene and ethyl acetate to give a mixture of 4a and 5a (the ratio changed with reaction time) as colorless syrup. The total yield of 4a and 5a was between 80% and 90%.

3-O-(2,3,4-tri-O-benzyl-α-D-glucopyranosyl)-1,2-di-O-
palmitoyl-sn-glycerols 8a and 8b: K 2 CO 3 (379 mg, 2.74 mmol) was added to the mixture of 4a and 5a (1 g, 1.83 mmol based on 4a) in CH 3 OH (20 mL) and stirred for 1 h at rt. The reaction mixture was neutralized, washed with water, dried, and concentrated. The residue was dried under reduced pressure and directly subjected to the next reaction. A mixture of caesium palmitate (2.7 g, 7.3 mmol) in DMF (40 mL) was heated at 100-110 °C, to which the DMF solution of the above residue was added slowly. The reaction mixture was stirred for 2 h at 110 °C, cooled to rt, and then filtered through a pad of Celite powder with ethyl acetate. The filtrate was washed with saturated aq. NaCl solution, dried, and concentrated. The residue was purified by silica gel column chromatography to give 6a as a colorless syrup (830 mg, 60% yield). To a solution of 6a (300 mg, 0.39 mmol) in pyridine (20 mL), TBDMS chloride (107 mg, 0.71 mmol) and 4-N,N-dimethylaminopyridine (cat.) were added. The reaction mixture was stirred for 12 h at rt, treated with methanol (2 mL) for 3 h and concentrated. The residue was purified by silica gel column chromatography in a mixture of toluene and ethyl acetate. The main product was dissolved in pyridine (20 mL) and then reacted with palmitoyl chloride (162 mg, 0.59 mmol) for 3 h at rt. The reaction mixture was treated with methanol (2 mL) and then concentrated with toluene. The residue was dissolved in a mixture of CH 3 OH and CH 2 Cl 2 (1:1, 20 mL) and treated with trifluoroacetic acid (1 mL) for 2 h at rt. After concentration, the residue was purified by silica gel column chromatography in a mixture of toluene and ethyl acetate to give 8a as a white waxy solid