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Molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-HT) to the human serotonin transporter (hSERT). Homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from Aquifex aeolicus, as the template and three slightly different sequence alignments. Induced fit docking of 5-HT into hSERT homology models resulted in two different binding modes. Both show a salt bridge between Asp98 and the charged primary amine of 5-HT, and both have the 5-HT C6 position of the indole ring pointing toward Ala173. The difference between the two orientations of 5-HT is an enantiofacial discrimination of the indole ring, resulting in the 5-hydroxyl group of 5-HT being vicinal to either Ser438/Thr439 or Ala169/Ile172/Ala173. To assess the binding experimentally, binding affinities for 5-HT and 17 analogues toward wild type and 13 single point mutants of hSERT were measured using an approach termed paired mutant-ligand analogue complementation (PaMLAC). The proposed ligand-protein interaction was systematically examined by disrupting it through site-directed mutagenesis and re-establishing another interaction via a ligand analogue matching the mutated residue, thereby minimizing the risk of identifying indirect effects. The interactions between Asp98 and the primary amine of 5-HT and the interaction between the C6-position of 5-HT and hSERT position 173 was confirmed using PaMLAC. The measured binding affinities of various mutants and 5-HT analogues allowed for a distinction between the two proposed binding modes of 5-HT and biochemically support the model for 5-HT binding in hSERT where the 5-hydroxyl group is in close proximity to Thr439.

An evaluation of whether the well-known deactivating effect of a 4,6-acetal protection group on glycosyl transfer is caused by torsional or an electronic effect from fixation of the 6-OH in the tg conformation was made. Two conformationally locked probe molecules, 2,4-dinitrophenyl 4,8-anhydro-7-deoxy-2,3,6-tri-O-methyl-beta-D-glycero-D-gluco-octopyranoside (18R) and the L-glycero-D-gluco isomer (18S), were prepared, and their rate of hydrolysis was compared to that of the flexible 2,4-dinitrophenyl 2,3,4,6-tetra-O-methyl-beta-D-glucopyranoside (21) and the locked 2,4-dinitrophenyl 4,6-O-methylidene-2,3-di-O-methyl-beta-D-glucopyranoside (26). The rate of hydrolysis at pH 6.5 was 21 > 18R > 18S > 26, which showed that the deactivating effect of the 4,6-methylene group is partially torsional and partially electronic. A comparison of the rate of acidic hydrolysis of the corresponding methyl alpha-glycosides likewise showed that the probe molecules 17S and 17R hydrolyzed significantly slower than methyl tetra-O-methyl-glucoside 19, confirming a deactivating effect of locking the saccharide in the (4)C(1) conformation. The experiments showed that the hydroxymethyl rotamers deactivate the rate of glycoside hydrolysis in the order tg >> gt > gg.

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Glycosyl donors protected with bulky silyl protective groups (tert-butyldimethylsilyl, TBS), on the 2-, 3-, and 4-OH groups were found to have superior reactivity compared with benzylated thioglucosides. The enhanced reactivity is explained by the stereoelectronic effects associated with the conformational change induced by the silylation. A TBS silylated thioglucoside donor has axial OR groups, whereas a benzylated thioglucoside has equatorial OR groups, leading to much more favorable charge-dipole interactions in the transition state. This concept could be used to create "super armed" glucosyl, mannosyl, rhamnosyl, and galactosyl donors, which could cross-couple with the armed acceptors, phenyl 2,3,4-tri-O-benzyl-beta-D-thioglucoside or phenyl 2,3,6-tri-O-benzyl-beta-D-thioglucoside, to give the corresponding armed disaccharides in good to excellent yields.