C-2 auxiliaries for stereoselective glycosylation based on common additive functional groups.

The stereoselective introduction of the glycosidic bond is one of the main challenges in chemical oligosaccharide synthesis. Stereoselective glycosylation can be achieved using neighbouring group participation of a C-2 auxiliary or using additives, for example. Both methods aim to generate a defined reactive intermediate that reacts in a stereoselective manner with alcohol nucleophiles. This inspired us to develop new C-2 auxiliaries based on commonly used additive functionalities such as ethers, phosphine oxides and tertiary amides. Good 1,2-trans-selectivity was observed for the phosphine oxide and amide-based auxiliaries expanding the toolbox with new auxiliaries for stereoselective glycosylation reactions.


Introduction
Carbohydrates play an essential role in biological systems. A major challenge in relating carbohydrate structure to function is the limited availability of structurally well-defined oligosaccharides and glycoconjugates. Such well-defined oligosaccharides can be obtained by chemical synthesis. The major challenge in oligosaccharide synthesis is the stereoselective introduction of the glycosidic bonds. 1,2 Stereoselective glycosylation can be achieved using neighbouring group participation (NGP) of a C-2 auxiliary or using additives, for example. Both methods aim to generate a defined reactive intermediate that reacts in a stereoselective manner with alcohol nucleophiles (Fig. 1A). For example, NGP of C-2 auxiliaries such as esters, picolyl ethers, 3 aryl nitriles 4 and ethers 5 mainly leads to 1,2trans-glycosides, whilst other C-2 auxiliaries based on esters, thioethers [6][7][8][9][10][11] and selenoethers 12 mainly lead to 1,2-cis-glycosides. Alternatively, the use of additives for stereoselective glycosylations requires a non-assisting functionality at C-2. 13 A Lewis base additive, such as nitriles, 14 ethers, 15,16 sulfides, 17 phosphine oxides, [18][19][20] iodide based reagents 21,22 and (form) amides, 19,20,23 is added to stabilize the glycosyl cation and introduce facial selectivity in the subsequent nucleophilic displacement by the glycosyl acceptor. Inspired by both these approaches we set out to develop new C-2 auxiliaries based on recently reported additives. To this end, new C-2 auxiliaries based on linear and cyclic ethers, phosphine oxides, and amide functionalities were prepared (Fig. 1B). Their glycosylation properties were established with a number of glycosyl acceptors. The ether-based auxiliaries showed very modest stereoselectivity whilst the phosphine oxide and amide based auxiliaries lead to the stereoselective formation of 1,2-trans glycosides.
To identify reaction intermediates for gluco-type donors 11 and 13, variable temperature (VT) NMR experiments were performed. Glycosyl donor 11 was reacted with Ph 2 SO and Tf 2 O in presence of TTBP at −80°C in CD 2 Cl 2 (Fig. 2). In a control experiment using glycosyl donor 11 and this promotor system (Tf 2 O, Ph 2 SO) no significant changes in yields or selectivity were observed indicating that the promotor system does not influence the glycosylation outcome. Directly after the addition of Tf 2 O the α-iminium intermediate (11α) was formed ( Fig. 2b-f ). In addition, a small amount of β-iminium ion (11β) was observed (α/β = 5/1). The ratio of intermediate 11α and 11β did not change even upon heating to room temperature and remained stable at this temperature (Fig. 2d).
Additionally, we performed VT NMR experiments under the same conditions for glycosyl donor 13 starting at −20°C ( Fig. 3a and c). After addition of Tf 2 O, the expected α-(13α) and β-phosphonium ions (13β) were observed in a α/β-ratio of 5/2 (Fig. 3). Both reaction intermediates were stable at 10°C and decomposed slowly at room temperature. Again, the ratio of 13α and 13β did not change upon heating. Based on these VT NMR results, the α-intermediates predominate for both the amide and phosphine oxide auxiliaries and may be reactive  a α/β ratios were determined by NMR spectroscopy 29,30 of the crude reaction mixture. b Isolated yields. c α/β ratios were determined by NMR spectroscopy after removal of the acceptor residues by silica gel flash column chromatography (30 to 80% EtOAc in n-heptane).
intermediates with strong nucleophiles. However, the erosion of stereoselectivity when glycosylating with secondary and tertiary alcohols suggests that other reactive intermediates such as the oxocarbenium ion may be responsible for disaccharide formation in these cases. Finally, we explored the removal of amide auxiliaries. Using disaccharide 50 as a model substrate, the tertiary amide was hydrolysed to yield the carboxylic acid. 31,32 After a simple workup the carboxylic acid was converted to the acyl azide, heated to form the isocyanate and reacted with t-BuOH at 100°C to yield the Boc-protected amine. After a short workup the Boc group was removed and the amine was subsequently treated with 1.0 M NaOH in which it was eliminated to yield the unprotected 2-OH (58) in 50% overall yield from 50 (Scheme 1). 33 In conclusion, we prepared a series glycosyl donor containing new C-2 auxiliaries based on commonly used additives for stereoselective glycosylation. The ether based auxiliaries gave rather unselective glycosylation reactions whilst tertiary amides and our phosphine oxide based auxiliaries showed good to absolute β-selectivity with reactive glycosyl acceptors. VT-NMR experiments confirmed the formation of reaction intermediates resulting from NGP of the C-2 auxiliaries. Potentially, chiral auxiliaries based on these functional groups can be developed to obtain even better stereoselectivity.

Experimental section
General conditions 1 H and 13 C NMR spectra were recorded on a Bruker 400 or 500 MHz spectrometer. Chemical shifts are reported in parts per million ( ppm) relative to tetramethylsilane (TMS) or residual solvents as the internal standard. NMR data is presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet and/or multiple resonances), coupling constant ( J) in hertz (Hz), integration. All NMR signals were assigned on the basis of 1 H NMR, 13 C NMR, 31 P NMR, COSY, HSQC, and TOCSY experiments. NMR data is presented for the major anomer. Mass spectra were recorded on an JEOL AccuTOF CS JMS-T100CS mass spectrometer. Automatic flash column chromatography was performed using Biotage Isolera Spektra One, using SNAP cartridges (Biotage, 30-100 μm, 60 Å), 4-50 g. TLC analysis was conducted on silica gel F254 (Merck KGaA) with detection by UV absorption (254 nm) where applicable; by spraying with 10% sulfuric acid in methanol followed by charring at ≈300°C or by spraying with KMnO 4 stain consisting of (0.06 M KMnO 4 , 0.5 M K 2 CO 3 and 0.02 M NaOH in water) after gently heating of the plate. DCM, THF, and toluene were freshly distilled. Molecular sieves (4 Å) were flame-activated under a vacuum prior to use. All inert reactions were carried out under an argon atmosphere using flame-dried flasks.
General procedure A 5.0 grams (0.035 mol, 1.0 eq.) D-glucal or D-galactal was dissolved in dry DMF (170 mL). 7.0 grams (0.18 mol, 5.0 eq.) NaH (60% dispersion in paraffin oil) was added on ice. The reaction was stirred for 15 minutes and 21 mL (0.18 mol, 5.0 eq.) benzyl bromide was added. The reaction was stirred at room temperature until TLC indicated full consumption of starting material (16 hours). The reaction was quenched with methanol and the solution was concentrated in vacuo. The resulting oil was taken up in ethyl acetate and washed with water (3 × 100 mL) and brine (1 × 100 mL). The organic layer was dried over MgSO 4 (anhydrous), filtrated and evaporated in vacuo to result the crude product. The benzylated products were obtained by purification of the crude product through silica gel flash column chromatography.

General procedure B
To a cooled (0°C) solution of 4.0 grams (9.6 mmol, 1.0 eq.) benzylated glycal in DCM (40 mL) were added acetone (4 mL) and saturated aqueous NaHCO 3 (68 mL). The mixture was  stirred vigorously, and a solution of oxone (19.2 mmol, 2 eq.) in H 2 O (24 mL) was added dropwise over 15 min. The mixture was stirred vigorously at 0°C for 30 min and then at rt until TLC indicated consumption of the starting material. The organic phase was separated, and the aqueous phase was extracted with DCM (2 × 40 mL). The combined organic phases were dried over Na 2 SO 4 and concentrated in vacuo. The crude mixture was dissolved in dry THF (80 mL). The solution was cooled to −78°C. MS (4 Å) and 2.2 grams (15 mmol, 1.6 eq.) sodium thiophenolate (90%) were added under an inert atmosphere. 1.0 mL (0.096 mmol, 0.1 eq.) ZnCl 2 (1.0 M in Et 2 O) was added and the mixture was stirred for 3 days allowing it to warm up to room temperature. An aqueous solution of 1.0 M NaOH (80 mL) was added to quench the reaction. The mixture was filtrated and the organic layer was separated from the aqueous layer. The organic layer was washed with brine (80 mL), dried over Na 2 SO 4 (anhydrous), filtrated and evaporated in vacuo. The crude product was purified with silica gel flash column chromatography to obtain the pure product. If the product was strongly coloured, the product was dissolved in DCM, norrit was added, filtrated over Celite and evaporated in vacuo to yield the product.

General procedure C
An anomeric thioether with free 2-OH (1 or 2, 1.0 eq.) was dissolved in dry DMF (0. 1 M) under an inert atmosphere at 0°C. 60% NaH dispersion in paraffin oil was added (2.0 eq. for the preparation of 11-14 and 34-39, 4.0 eq. for 3-10). After stirring for 15 minutes, ice was removed and the corresponding bromide or tosylate (4.0 eq.) was added. The reaction was stirred for overnight after which the reaction was quenched with methanol and the solvent was evaporated in vacuo. The donors were obtained by purification through silica gel flash column chromatography.

General procedure D
The corresponding glycosyl donor (1.0 eq.) and the corresponding glycosyl acceptor (2.0 eq.) were dissolved in dry DCM (0.02 M and 0.04 M respectively). MS (4 Å) were added and the mixture was cooled to −15°C. NIS (1.1 eq.) was added followed by the addition of a catalytic amount TfOH (0.1 eq.). The reaction was stirred for 2 h allowing it to slowly reach room temperature. The reaction was quenched with TEA and taken up in EtOAc (20 mL). The solution was filtrated, washed with aqueous thiosulfate solution (10%, 20 mL) and washed with brine (20 mL). The organic layer was dried over MgSO 4 (anhydrous), filtrated and the solvent was evaporated in vacuo to yield the crude product. The crude product was dissolved in CDCl 3 and analysed by quantitative HSQC to determine the selectivity. After analysis the crude product was purified by flash column chromatography to obtain the product.