Synthesis of photoaffinity probes [2 ′-iodo-4 ′-(3 ′′-trifluoromethyldiazirinyl)phenoxy]- d-glucopyranoside and [(4 ′-benzoyl)phenoxy]- d-glucopyranoside for the identification of sugar-binding and phlorizin-binding sites in the sodium/ d-glucose cotransporter protein

Abstract

In this paper we describe the synthesis and photochemical and biochemical properties of two new photoaffinity probes designed for studies on the structure–function relationship of the sodium d-glucose cotransporter (SGLT1). The two probes are [2 ′-iodo-4 ′-(3 ′′-trifluoromethyldiazirinyl)phenoxy]- d-glucopyranoside (TIPDG), a mimic for the phenyl glucopyranoside arbutin which is transported by SGLT1 with a very high affinity, and [(4 ′-benzoyl)phenoxy]- d-glucopyranoside (BzG), a model compound for phlorizin, the most potent competitive inhibitor of sugar translocation by SGLT1. Both photoaffinity probes TIPDG ( λ max=358 nm) and BzG ( λ max=293 nm) can be activated at 350–360 nm, avoiding protein-damaging wavelengths. In inhibitor studies on sodium-dependent d-glucose uptake into rabbit intestinal brush border membrane vesicles TIPDG and BzG showed a fully competitive inhibition with regard to the sugar with respective K i values of 22 ± 5 μM for TIPDG and 12 ± 2 μM for BzG. These K i values are comparable to those of their parent compounds arbutin (25 ± 6 μM) and phlorizin (8 ± 1 μM). To further test the potential of TIPDG and BzG as photoaffinity probes, truncated loop 13 protein, supposed to be part of the substrate recognition site of SGLT1, was exposed to TIPDG and BzG in solution. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis demonstrated that TIPDG and BzG successfully labeled the protein. These preliminary results suggest that both photoaffinity probes are promising tools for the study of the structure–function relationship of SGLT1 and other SGLT1 family transporter proteins.