Direct Evidence for Remote Participation in Galactose Building Blocks during Glycosylations Revealed by Cryogenic Vibrational Spectroscopy.

The stereoselective formation of 1,2- cis -glycosidic bonds is challenging. However, 1,2- cis -selectivity can be induced by remote participation of C4 or C6 ester groups. Reactions involving remote participation are believed to proceed via a key ionic intermediate, the glycosyl cation. Although mechanistic pathways were postulated many years ago, the structure of the reaction intermediates remained elusive due to their short-lived nature. Here, we unravel the structure of glycosyl cations involved in remote participation reactions via cryogenic vibrational spectroscopy and first principles theory. Acetyl groups at C4 ensure α-selective galactosylations by forming a covalent bond to the anomeric carbon in dioxolenium-type ions. Unexpectedly, also benzyl ether protecting groups can engage in remote participation and promote the stereoselective formation of 1,2- cis -glycosidic bonds.


Calculated Low Energy Structures
: Calculated structure of glycosyl cation A generated from the 4,6Ac building block. Hydrogens are omitted for clarity.

Preparation of 6-O-acetyl-2,3,4-tri-O-benzyl-α-D-galactopyranosyl trichloroacetimidate (S10α)
To a solution S8 4 (279 mg, 0.52 mmol) in THF (3 mL) and water (0.3 mL) were added N-iodosuccinimide (87.7 mg, 0.39 mmol) and 1M HCl 2 drops, and then stirred for 3 h at room temperature. The reaction mixture was quenched with with sat. aq. NaHCO3 solution (10 mL) and DCM (10 mL). The organic layer was extracted with DCM (2 × 10 mL) and washed with brine (10 mL). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure for column chromatography purification (Elution: n-hexane/EtOAc = 6/1 to 2/1) and obtained as an inseparable α/β mixture S8  Figure S12: Reactions of galactose building blocks to assess the impact of different protecting group combinations (Bn=benzyl, Ac=acetyl) on the stereochemical outcome of the glycosylation reaction. All reactions including Bn data were performed by following same methodology given in ref. 5.

Analysis Section:
The reactions were monitored using HPLC. The HPLC system used was a Knauer Platin Blue system, equipped with a UV detector (254 nm). The column used was YMC-Pack Diol Normal Phase diol column (DN12S05-2546WT) with particle size of 5 µm. The column has an I.D. of 4.6 mm and length of 250 mm. The column was housed inside a column oven, and was maintained at 20 °C for all analysis. The mobile phase was gradient mixture of HPLC grade ethyl acetate and hexane, which was pumped with a constant flowrate of 1 mL/min. The gradient system of the mobile phase was developed and programmed into the HPLC.             After completion of all GA runs, all structures generated for each carbocation were merged and clustered using root-mean square deviation (RMSD) metric for distances between heavy atoms. The tight RMSD=0.1 Å cutoff was selected to judge structural similarity. The two-fold symmetry of benzyl rings were included in similarity comparison. The RMSD calculations were performed in mdtraj-1.9.1 python module [6] , while further hierarchical clustering was done with scipy-1.1.0 python module. Number of unique structures obtained for each cation is shown in Table 2. Next, we measured a distance between anomeric carbon C1 and the respective oxygen in the acetyl group and plotted the relative energy as a function of this distance (Fig 1).
The potential energy of each molecule was finally computed using Resolution-of-Identity [8] MP2 method, extrapolated to the complete basis set. These calculations were performed using ORCA-4.1 software [9] The extrapolation was done using two-point extrapolation [10] with def2-TZVPP and def2-QZVPP basis sets and auxiliary def2-QZVPP/C basis set for RI. Our previous calculations on monosaccharides showed that RI yields virtually identical energies to those from canonical MP2 calculations. Grid5 settings and tight SCF convergence were requested to obtain full convergence. The reported conformational energies are corrected by free-energy contributions at 78K derived from harmonic vibrational calculations performed described in previous paragraph. Figure S56. Energy hierarchys of the initial sampling for galactosyl cations generated from 4,6Ac (left) and 4Ac (right) precursor. The bold markers indicate structures bearing same ring pucker as the lowest energy structure. Figure S57. Energy hierarchies of the initial sampling for galactosyl cations generated from 6Ac (left) and Bn (right) precursor. The bold markers indicate structures bearing same ring pucker as the lowest energy structure.

Supplementary Tab. 3
List of conformations of 46Ac reoptimized with PBE0+D3 functional: conformers' id labels, assigned ring puckers, distances between anomeric carbon and oxygen, energies computed at PBE0+D3/6-311+G(d,p) level of theory, harmonic free energy at 78K, RI-MP2 single-point energy extrapolated to the complete basis set from def2-TZVPP and def2-QZVPP basis sets and relative MP2/CBS energy corrected with harmonic free energy from DFT in kcal mol -1 . Conformers analysed in the paper are indicated with a label in parenthesis. The PBE0+D3/6-311+G(d,p) energies labelled with an asterisk (*) have been computed using Gaussian16, RevA.03 [11] defaults. Tab. 4 List of conformations of 4Ac reoptimized with PBE0+D3 functional: conformers' id labels, assigned ring puckers, distances between anomeric carbon and oxygen, energies computed at PBE0+D3/6-311+G(d,p) level of theory, harmonic free energy at 78K, RI-MP2 single-point energy extrapolated to the complete basis set from def2-TZVPP and def2-QZVPP basis sets and relative MP2/CBS energy corrected with harmonic free energy from DFT in kcal mol -1 . Conformers analysed in the paper are indicated with a label in parenthesis.  Figure S58. Energy hierarchies of the reoptimized structures for galactosyl cations generated from 4,6Ac (left) and 4Ac (right) precursor. The bold markers indicate structures bearing same ring pucker as the lowest energy structure. In case of the 4,6Ac cation, black crosses indicate dioxolenium ions with C6-participation, whereas red crosses indicate oxonium structures with two unbound acetyl groups. Figure S59. Energy hierarchies of the reoptimized structures for galactosyl cations generated from 6Ac (left) and Bn (right) precursor. The bold markers indicate structures bearing same ring pucker as the lowest energy structure. Figure S60. Different tautomers of Ac6. The numbers indicate relative free energy at 300K with respect to the conformer G. The structures are shown in the right, hydrogens at the anomeric carbon and at the carbon the they are transferred to are sown with white balls. The labels underneath represent from where and where to they were transferred. C2H means the carbon 2 in the pyranose ring, the BnO(CX) means a methylene carbon in the respective benzyl group. Although some of the structures have lower energy, none of them explains three bands at 1300 cm -1 and 1600 cm -1 .