Chemical synthesis of amphipathic glycosides
Dodecyl lactoside is the standard used to investigate glycosylation in cells. We prepared dodecyl β-acetyl lactoside by reacting acetyl lactosyl imidate with dodecane-1-ol using BF3·Et2O (scheme ). The dodecyl β-acetyl lactoside was then deacetylated with NaOMe in MeOH, with the amphipathic glycoside, dodecyl β-lactoside, obtained in a 51.6% yield (2 steps).
Synthesis of amphipathic lactosides. i) NaN3, DMSO, 80°C. ii) BF3·Et2O, ClCH2CH2Cl, 0°C. iii) NaOMe, MeOH, r.t.
We also synthesized 12-azidododecyl lactoside. 12-Bromo dodecane-1-ol was reacted with sodium azide in DMSO yielding 12-azidododecane-1-ol, which was then glycosylated with lactosyl imidate by the same procedure, before being deacetylated to give 12-azido dodecyl lactoside in 61.3% yield (3 steps). The product has the azido group at the ω-position of the alkyl chain, which can be easily converted to an amino group for further utilization.
Glycosylation of dodecyl lactoside by Vero cells
To investigate how the amphiphatic lactoside is glycosylated, dodecyl lactoside was incubated with Vero cells. After incubation, the glycolipids – which were collected from the culture medium and cell homogenates – were analyzed by HPTLC. As shown in Figure , three new bands, which were not obtained in the control, were observed. It was thought likely that these corresponded to Gb3, Gb4 and GM3 type oligosaccharides, owing to these being known to occur naturally in Vero cells [12
Figure 1 HPTLC results. (A) Glycolipids after incubation of Vero cells with dodecyl lactoside. Lane 1, glycolipids extracted from cell homogenate; Lane 2, glycolipids extracted from medium; Lane 3, dodecyl lactoside. (B) Glycolipids after incubation of Vero cells (more ...)
To determine the structure of the glycosylated sugar moiety, the glycosylated lactosides were scraped from the HPTLC plates, collected and extracted. Owing to the small quantities of collected glycosides NMR spectra could not be measured; analysis was thus carried out by ESI-MS. The mass spectra results showed peaks at m/z 533.4, 695.1, 898.5 and 987.7 corresponding to [dodecyl lactoside + Na]+
, [dodecyl Gb3 + Na]+
, [dodecyl Gb4 + Na]+
and [dodecyl GM3 + Na]+
, respectively. These results are consistent with the glycosides anticipated from HPTLC analyses. The three new bands were thus assumed to be analogues of Gb3 (galactosylated lactoside derivative), Gb4 and GM3. These oligosaccharides were produced by only one cell type, therefore, different kinds of oligosaccharides can be produced using various cells. This method of administering the glycosides to the cells is also effective in biocombinatorial synthesis for the construction of an oligosaccharide library [16
In the same manner, 12-azidododecyl lactoside was incubated with Vero cells and the glycosides in the culture medium were developed on HPTLC. From HPTLC analysis, three new bands were observed (at same positions observed for dodecyl lactoside) and corresponding to Gb3, Gb4 and GM3.
Influence of seeded cell number
To evaluate the influence of seeded cell number on the yield of glycosylated lactoside, 2 × 106, 5 × 106 and 10 × 106 Vero cells were seeded on 100 mm dishes. The seeded cells were incubated for 24 h in a medium containing 10% FBS before being incubated in a medium containing 50 μM of 12-azidododecyl lactoside for a further 48 h. Quantification of the amount of glycosylated product with a densitometer revealed that the relative ratio of the absorbances of the galactosylated lactoside bands was 1.00 (2 × 106 cells): 1.39 (5 × 106 cells): 1.74 (10 × 106 cells) as shown in Figure . Therefore, the quantity of glycosylated product increased with increasing seeded cell number, with 2 × 106 cells most efficient for the quantity of product obtained per-cell.
Influence of cell culture conditions. Relative ratio of the amount of product in different (A) seeded cell numbers; (B) incubation times; (C) lactoside concentrations; and (D) medium volumes.
Influence of the incubation time
To evaluate the influence of incubation time on the yield of glycosylated lactoside, 2 × 106 Vero cells were seeded on 100 mm dishes. The cells were incubated in a medium containing 10% FBS for 24 h, before further incubation for 48, 72 and 96 h in a medium containing 50 μM 12-azidododecyl lactoside. Analysis of the amount of glycosylated product with a densitometer revealed that the relative ratio of the absorbances of the galactosylated lactoside bands was 1.00 (48 h): 1.78 (72 h): 1.84 (96 h) as shown in Figure . An incubation time of 72 h was most efficient in terms of the amount of product obtained as a function of time.
Influence of the concentration of 12-azidododecyl lactoside
To evaluate the influence of the concentration of 12-azido dodecyl lactoside on the yield of glycosylated lactoside, 2 × 106
Vero cells were seeded on 100 mm dishes and incubated in a medium containing 10% FBS for 24 h. Subsequently, the dishes were incubated in media containing 50, 100, 150 and 200 μM of 12-azidododecyl lactoside for 48 h. Quantification of the amount of glycosylated product with a densitometer revealed that the relative ratio of the absorbances of galactosylated lactoside band was 1.00 (50 μM): 1.46 (100 μM): 1.54 (150 μM): 1.17 (200 μM) as shown in Figure . The largest amount of galactosylated lactoside was obtained from the incubation with 150 μM of lactoside, while cytotoxicity was observed for the 200 μM medium (See additional file 1
: Figure 4). The medium containing 50 μM of lactoside was seen to be the most efficient in terms of the yield of galactosylated lactoside.
Influence of medium volume
To evaluate the influence of media volume on the yield of glycosylated lactoside, 5 × 106 Vero cells were seeded on 100 mm dishes. The cells were incubated in a medium containing 10% FBS for 24 h, before incubation for 48 h in 7.0, 10.5, 14 and 35 ml of media containing 50 μM 12-azidododecyl lactoside. Analysis with a densitometer of the quantity of glycosylated product revealed that the relative ratio of the absorbances of galactosylated lactoside bands was 1.00 (7 ml): 1.59 (10.5 ml): 1.42 (14 ml): 1.63 (35 ml) as shown in Figure . Even if the volume of the medium were increased, the yields of galactosylated lactoside were almost similar to those obtained from 10.5 ml. Therefore, the 10.5 ml medium was deemed most suitable for the production of glycoside.
The quantity of galactosylated lactoside obtained under optimum conditions
We selected the following conditions: seeded cell number of 5 × 106
or 10 × 106
cells; 72 h incubation period; lactoside concentration of 100 or 150 μM; and medium volume of 10.5 ml. Under these conditions, Vero cells were seeded on 100 mm dishes and incubated in media containing lactoside (See additional file 2
: Figure 5). The results showed that the highest yield of galactosylated lactoside obtained was 87.9 μg/dish (11.7 %). From the results, the optimum set of conditions were seen to be a 72 h incubation period, using 5 × 106
cells in the presence of 100 μM lactoside and 10.5 ml of medium solution.
Repeated administration and harvest
After the solution containing galactosylated lactoside derivative was harvested from the medium of Vero cells, the lactoside-containing medium was successively added and incubated. The concentration of lactoside in the media was 100 μM, with the administration and harvest carried out every 24, 48 or 72 h. The time period of the cell culture was twelve days, with the amounts of galactosylated lactoside from each of harvested media quantified using densitometer (Figure ).
Galactosylated lactoside (Gb3) produced after repeated administration of 12-azidododecyl lactoside in Vero cells with harvest of incubated medium at repeated time periods.
In the case of the 24, 48 and 72 h cycles, the total quantities of galactosylated lactoside obtained were 430 μg (602 nmol, 5.21%), 371 μg (520 nmol, 8.25%) and 347 μg (486 nmol, 11.6%), respectively.
These results indicated that Vero cells are suitable for continuous culture over long, repeated time periods and are useful for the mass production of glycosides.
Large-scale production of galactosylated lactoside
For the production of large amounts of galactosylated lactoside, Vero cells were seeded on a microcarrier in a spinner flask and cultured for 2 weeks [17
]. The concentration of lactoside was 100 μM and repetition cycles lasted 48 or 72 h. The results showed that a 48 h repetition time frame (rather than 72 h) was preferable, with Gb3 quantities obtained being 4.7 μg/ml 3.3 μg/ml for 48 and 72 h, respectively. Finally, Vero cells on a microcarrier in 4 litre spinner flask were incubated for 48 h, with the administration and harvest repeated 5 times. The total amount of galactosylated lactoside obtained was 30.2 mg (42.4 μmol, 1
H NMR spectrum: H-1, J1,2
= 7.8; H-1', J1',2
' = 7.2; H-1", J1",2"