Acceptor-influenced and donor-tuned base-promoted glycosylation

Base-promoted glycosylation is a recently established stereoselective and regioselective approach for the assembly of di- and oligosaccharides by using partially protected acceptors and glycosyl halide donors. Initial studies were performed on partially methylated acceptor and donor moieties as a model system in order to analyze the key principles of oxyanion reactivities. In this work, extended studies on base-promoted glycosylation are presented by using benzyl protective groups in view of preparative applications. Emphases are placed on the influence of the acceptor anomeric configuration and donor reactivities.


Introduction
For the assembly of oligosaccharides the complex and challenging control of regio-and stereochemistry has to be solved. Various contributions have facilitated enormously the access to complex oligosaccharide structures so far; however, regioselective and stereoselective glycosylations remain difficult and alternative concepts are welcome, and should be considered and studied.
Recently regioselective and β-stereospecific glycosylations employing saccharide oxyanions have been presented. Initial studies on base-promoted glycosylations were elaborated on partially methylated glucopyranosides as model systems. This systematic approach allowed determination of the preferred positions for glycosylation and the establishment of an oxyanion reactivity scale. In addition to studies with different base promoters and donors, the first successful conversions with benzylated acceptor and donor moieties were reported [1][2][3].
This novel glycosylation method could substantially reduce the need to apply protecting-group chemistry. Also, the β-specificity and base-promoted activation without any heavy-metal salt or Lewis acid are useful for synthetic applications. To develop the synthetic potential and evolve this method into an applicable alternative pathway towards di-, tri-or oligosaccharides, more research needs to be done.
So far, glycosylation reactions have been conducted by employing methyl α-glycopyranoside acceptors. Therefore, it was of particular interest to test the base-promoted glycosylation methodology with methyl β-glycopyranosides. Hence monobenzylated methyl α-and β-D-glycopyranosides were synthesized, then fucosylated by employing the base-promoted glycosylation methodology, and analysed with respect to the absolute and relative yields.
In addition, donor reactivity in base-promoted glycosylation reactions was investigated by employing partially benzylated methyl α-and β-D-glycopyranosides and donor halide mixtures.

Results and Discussion
To answer the questions above, monobenzylated methyl α-and β-D-gluco-and galactopyranosides 1-6 were selected as acceptor moieties ( Figure 1) and synthesized by employing standard protecting-group chemistry. After base activation of the acceptor hydroxy groups, glycosylation was performed with the perbenzylated glycopyranosyl chlorides 7-9 ( Figure 2).
The results of the base-promoted glycosylation of monobenzylated methyl α-and β-D-glycopyranosides with fucopyranosyl chloride 7 are summarized in Table 1 only the β-(1→6)-linked disaccharide 37 was found. Thus, in all α-methyl glycopyranoside acceptors 1, 3 and 5, successful glycosidic bond formations with high regioselectivity towards position 6 were observed. These findings can be understood by the presence of adjacent hydroxy groups on acceptors 1, 3 and 5, such as 4,6-diol and/or 3,4,6-triol structures, respectively. After deprotonation the negative charge is dispersed by hydrogen bonding of unreacted hydroxy groups and located predominantly at position 6 due to the higher acidity of OH-6 [3]. Additionally, the basicity of the oxyanions stabilized by hydrogen bonding is decreased in comparison to isolated oxyanions, and this favours glycosidic bond formation over elimination. Table 1: Glycosylation results for acceptors 1-6 with NaH as base and 2,3,4-tri-O-methyl-α-L-fucopyranosyl chloride (Fuc-Cl, 7) as donor. a

Acceptor
Products Surprisingly glycosylation of the corresponding β-glucopyranosides 2, 4, and 6 only showed conversion for the 3-O-benzylated glucopyranosyl derivative 4. Acceptors 2 and 6 did not react at all with the fucopyranosyl chloride 7. Obviously, the anomeric configuration in the acceptor structure has a noticeable effect on the glycosylation, resulting in lower oxyanion reactivity for methyl β-glycopyranosides. The considerable effect of the anomeric configuration on the reactivity of remote hydroxy groups may be rationalized by stereoelectronic effects [12]. Moreover, the lower reactivities of the methyl β-glycopyranosides may also be dependent on the decreased solubility of the partially deprotonated methyl β-glycospyranosides in the solvent used.
The results of base-promoted glycosylations with donor mixtures are shown in Table 2   equimolar mixtures of the perbenzylated fuco-and glucopyranosyldonors 7 and 9 gave just the β-(1→6)-fucosylated products 35 and 36 in corresponding yields of 15% and 14%. Furthermore, a mixture of galacto-and glucopyranosyl donors 8 and 9 was employed, but this did not show any reaction at all. In all cases, disaccharide yields from donor mixtures were significantly lower; as rational of which, some unknown kind of interaction between the donors may be assumed.

Conclusion
These studies gave evidence that the configuration of the anomeric centre of acceptors has a decisive influence on the base-promoted glycosylation approach, but which so far cannot be explained. Several α-monobenzyl acceptors (1, 3 and 5) and their β counterparts (2, 4 and 6) were synthesized and coupled with the perbenzylated fucopyranosyl donor 7. Whereas all α-glycosidic acceptors gave disaccharides in moderate to acceptable yields, only the β-glycosidic acceptor 4 reacted, to give the dissacharide in much lower yield.
Furthermore, a comparison between the reactivities of several donor mixtures was established. For this purpose the perbenzylated donors 7-9 were synthesized and reacted in equimolar mixtures with acceptors 3 and 4. The fucopyranosyl donor 7 showed the highest reactivity, followed by the galactopyranosyl donor 8. The glucopyranosyl donor 9 showed no reaction at all. It turned out that the disaccharide yields from the mixtures were significantly lower, indicating unknown interactions between donors.
In cases with low yields or even no glycosylation products at all, elimination became the dominant reaction and led to the corresponding glycals. Hence, and based on the workup, the recovery of glycosyl halides was neither practicable nor achieved.

Experimental
General: All reagents were purchased from commercial sources and used as received. Sodium hydride (NaH) was used as a 60% suspension in paraffin. TLC was performed on Merck silica gel 60 F 254 plates. Compounds were detected by UV and/ or by treatment with EtOH/H 2 SO 4 (9:1) and subsequent heating. Column chromatography was performed with Merck/ Fluka silica gel 60 (230-400 mesh). Solvents for column chromatography were distilled prior to use. 1 H and 13 C NMR spectra were recorded with Bruker AMX-400 or Bruker AV-400 spectrometers (400 MHz for 1 H, 100 MHz for 13 C) and calibrated by using the solvent residual peak. In CDCl 3 TMS was used for calibration. Melting points were measured with an Apotec melting point apparatus. Optical rotations were obtained by using a Krüss Optronic P8000 polarimeter (589 nm, 25 °C). HRMS (ESI) were recorded with a Thermo Finnigan MAT 95XL mass spectrometer. MS (MALDI-TOF) were recorded with a Bruker Biflex II (positive reflection mode, matrix: 2,5dihydroxybenzoic acid). Relative yields of disaccharide mixtures were determined by integration of the signals in the 1 H NMR spectra of the anomeric or other well-separated protons. The abbreviation "dd~vt" stands for a double doublet that turns into a virtual triplet because of similar coupling constants. Physical and NMR data for 34 and 35 were published previously [1].
General Procedure: Base-promoted glycosylations: The acceptor molecule (1.0 mmol) was dissolved in dry dimethylformamide (2-4 mL), and sodium hydride (1.1-1.3 mmol for each hydroxy group; as a 60% suspension in paraffin) was added. After being stirred for one hour under argon at room temperature, the donor (1.0 mmol for each hydroxy group; donor-mixtures 0.5 mmol of each donor) dissolved in dry dimethylformamide (2-4 mL) was successively added to the reaction. After stirring for 4-100 h, methanol (2 mL) was added. The crude product was purified by flash column chromatography (petroleum ether/ethyl acetate, 2:1 v/v).