Glycosyl Photoaffinity Probes: Synthesis and Application

 One of the major events occurring at biological interfaces is the specific recognition of bioactive ligands by their receptor proteins. The method of photoaffinity labeling enables the direct probing of target protein through a covalent bond introduced between a ligand and its specific receptor (1).

The first step in photoaffinity labeling is the synthesis of photoreactive probes by attaching a photoreactive group on a ligand molecule such as a carbohydrate. Among photoreactive groups currently used, diazirine meets most of the criteria for an ideal photoreactive group. In addition to its super reactivity and the stability of cross-link, diazirines can be rapidly photolyzed at wavelengths beyond the UV absorbance region of proteins. Several biotinylated diazirines were developed as a non-radioisotopic solution to the laborious photoaffinity labeling routine (Figure). Recently, a biotinylated diazirine with an aminooxy group (R = CH2ONH2) was developed for the protection-free preparation of carbohydrate photoprobes from the micromole quantities of various oligosaccharides. By a single-step reaction, a biotinylated carbene-generating unit can be introduced to the reducing end of unprotected carbohydrates. By using the commercially available biotinyl diazirine (Seikagaku Corporation), a sequence of photoaffinity labeling experiments, from the probe synthesis to the detection of labeled protein, could be readily accomplished within one week (2).

Photoaffinity labeling can be applied at two levels (Figure). At the protein level, the cross-link is useful for the identification of target proteins by a SDS-PAGE analysis (bottom left). If the binding site analysis of the target protein is important, the photoaffinity labeling will give sequence information on binding site peptides (bottom right). Recent developments in mass spectroscopy provide an efficient way for the analysis of peptide sequences.

Major approaches currently used in the investigation of protein functional sites have their own advantages and limitations. The genetic approach, for example, provides a series of mutants for the structural analysis of functional sites. However, it is generally impossible to exclude potential conformational changes that may result in alterations of ligand binding processes. Thus, photoaffinity labeling is a reliable chemical method which should be considered as being complementary to, rather than in competition with other approaches. A biotinylated photoaffinity probe was applied in the analysis of acceptor binding-site within 1,4-galactosyltransferase. The photochemically introduced biotin tag harnesses the power of avidin-biotin technology for one-step purification of photolabeled peptides from the protein digest. The approach yielded, for the first time, information on the acceptor site peptides in this enzyme. A molecular docking study based on this fragment strongly suggests an active site carboxylate which could be involved in the galactosyl transfer step. The result also demonstrates that the combination of photoaffinity labeling, crystallography and 3D graphics can be a powerful solution for detailed structural analysis of ligand-protein complexes (3).
(Faculty of Pharmaceutical Sciences,
Toyama Medical and Pharmaceutical University)
References (1) Hatanaka Y.; Sadakane Y., Photoaffinity Labeling in Drug Discovery and Developments: Chemical Gateway for Entering Proteomic Frontier, Curr. Top. Med. Chem., 2, 271-288, 2002.
(2) Hatanaka, Y.; Kempin, U.; Park, J. -J., One-step Synthesis of Biotinyl Photoprobes from Unprotected Carbohydrates. J. Org. Chem., 65, 5639-5643, 2000.
(3) Hatanaka, Y.; Ishiguro, M.; Hashimoto, M.; Gastinel, L. N.; Nakagomi, K., A Model of Photoprobe Docking with 1,4-Galactosyltransferase Identify a Possible Carboxylate Involved in Glycosylation Steps. Bioorg. Med. Chem. Lett., 11, 411-413, 2001.
Apr. 1, 2003

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