Synthesis and Characterization of Photoaffinity Probes that Target the 5-HT3 Receptor

Abstract

Abstract:The 5-HT 3 receptor is one of several ion channels responsible for the transmission of nerve impulses in the peripheral and central nervous systems. Until now, it has been difficult to characterize transmembrane receptors with classical structural biology approaches like X-ray crystallography. The use of photoaffinity probes is an alternative approach to identify regions in the protein where small molecules bind. To this end, we present two photoaffinity probes based on granisetron, a well known antagonist of the 5-HT 3 receptor. These new probes show nanomolar binding affinity for the orthosteric binding site. In addition, we investigated their reactivity using irradiation experiments.

One‐Pot Synthesis of Diazirines and 15N2‐Diazirines from Ketones, Aldehydes and Derivatives: Development and Mechanistic Insight

“…which was successfully used by our group for the synthesis of diazirines from α‐amino acids; [13] however, it led to the formation of the expected diazirine 2 a in only 11% isolated yield (Table 1, Entry 1). With metal oxidants such as silver(I) oxide (Ag 2 O), or manganese(IV) oxide (MnO 2 ) [20] that were already reported to oxidize diaziridines into diazirines, ketoamide 1 a did not react and was recovered unchanged (entries 2 and 3). A similar observation was made with the use of t ‐BuOK under air, which has recently been reported as an effective strategy for the dehydrogenation of the NH−NH bond in particular for diazirine synthesis (Entry 4) [19a] .…”

Mapping the Orthosteric Binding Site of the Human 5-HT3 Receptor Using Photo-cross-linking Antagonists

“…Following our previous SAR study of granisetron and design of fluorescent granisetron probes, , we chose to link the photolabeling moieties to the indazole N -1 or C-7 or granatane N -9′ position of the antagonist core (Scheme ). Briefly, either 1 H -indazole amide 1 or 7-hydroxyindazole amide 7 , was alkylated with protected linkers, followed by deprotection and subsequent alkylation with benzophenone bromide 17 or diazirine benzyl bromide 19 or amidation with diazirine benzoic acid 20 . , For accessing the N -9′ modified probes 15 and 16 , Boc-protected granatane amine 12 was alkylated first with diazirine benzyl iodide 21 , , deprotected, coupled to 1 H -indazole-3-carboxylic acid, and finally N -1 alkylated.…”

“…For alternative predicted binding orientations of 4 and 10, see Figure S7. 39 Amine 3 35 (28.4 mg, 66 μmol) was dissolved in dry DMF/DCM 3:2 (5 mL), and then carboxylic acid 20 (15.3 mg, 66 μmol), HATU (50.4 mg, 133 μmol), and three drops of Et 3 N were added. The reaction mixture was stirred at RT overnight.…”

“…Briefly, either 1H-indazole amide 1 35 or 7-hydroxyindazole amide 7 31,35 were alkylated with protected linkers, followed by deprotection and subsequent alkylation with benzophenone bromide 17 36 or diazirine benzyl bromide 19, 37 or amidation with diazirine benzoic acid 20. 38,39 For accessing the N-9' modified probes 15 and 16, Boc-protected granatane amine 12 40 was alkylated first with diazirine benzyl iodide 21, 41,42 deprotected, coupled to 1H-indazole-3-carboxylic acid and finally N-1 alkylated.…”

The binding orientation of epibatidine at α7 nACh receptors

“…Our finding that the orientations of epibatidine in the AChBP co-crystal structures are broadly correct, and that 3SQ6 shows improved accessibility at the C-4′ position, suggests that there may still be opportunities to develop epibatidine-based probes with higher affinities for the α7 nACh receptor. In our previous work the development of similar probes for targeting 5-HT 3 receptors has enabled quantitative fluorescent measurements, receptor localisation and photo cross-linking to be performed, and shows the value of pursing their synthesis (Lochner and Thompson, 2015, Jack et al., 2014). …”