Synthesis and Biological Evaluation of Didemnin Photoaffinity Analogues Matthew D. Vera, a Amy J. Pfizenmayer, a Xiaobin Ding, a Deepika Ahuja, b Peter L. Toogood b and Madeleine M. Joullie´ a, * a DepartmentofChemistry,UniversityofPennsylvania,Philadelphia,PA19104-6323,USA b DepartmentofChemistry,UniversityofMichigan,AnnArbor,MI48109-1055,USA Received 21 March 2001; accepted 2 May 2001 Abstract—The synthesis of four benzophenone-containing analogues of the antiproliferative natural product didemnin B is pre- sented. In vitro protein biosynthesis inhibition potency and antitumor activity were evaluated. The results indicate that all four analogues are biologically active and could serve as photoaffinity reagents for the study of receptor-binding interactions of didemnins. These analogues could also be useful in studying antitumor effects of didemnins. # 2001 Elsevier Science Ltd. All rights reserved. The didemnin class of marine depsipeptides (1 and 2, Fig. 1) has attracted considerable interest resulting from the wide spectrum of biological activities exhibited by didemnin B, the initial lead congener. 1 3 Although didemnins have shown antiviral, immunosuppressive, and protein biosynthesis inhibition effects, the anti- proliferative and cytotoxic actions have led to clinical trials of some congeners against cancer. 4 The clinical efficacy of didemnins remains uncertain. Moreover, lit- tle is understood about the molecular basis of their biological activities. Two didemnin binding proteins, eukaryotic elongation factor 1a (EF-1a) 5 and palmitoyl protein thioesterase 1 (PPT1) 6 have been identified. While the mechanistic relevance of the interaction with PPT1 is unclear, 7 binding of didemnin B to EF-1a explains the protein biosynthesis inhibition effects. 8 Using a tritiated didem- nin B radioligand, we recently reported evidence show- ing that a ternary complex consisting of ribosome, didemnin B, and EF-1a serves to inhibit the EF-2-medi- ated translocation step of eukaryotic peptide elongation in vitro. 9,10 Although mechanistically interesting, the inhibition of protein biosynthesis by didemnins is not sufficient to explain the cellular apoptosis induced by these natural products. 11 Moreover, the interaction between didemnin B and its two binding proteins is not understood at any level of structural detail. In order to address this aspect of didemnin biochem- istry, we now report the synthesis and biological eval- uation of didemnin photoaffinity analogues. Before attempting to use any analogues for photoaffinity label- ing, we decided to address two separate questions. First, we wished to establish whether the presence of a pho- tophore in the side chain would be tolerated. Secondly, we wished to determine whether there is an optimal length of the linker between the photophore and the didemnin nucleus. Analogues 3–6 (Fig. 1) were designed to explore both issues. Photoaffinity analogue candidates 3–6 are formally didemnin A derivatives containing the 4-benzoylbenzoic acid moiety. We chose this type of benzophenone deri- vative as the photoreactive group because of its multiple advantages in photoaffinity labeling. 12,13 Benzophe- nones are easily manipulated in ambient light, but are photoactivated at about 350 nm, a wavelength which will not damage the target protein. The activated diradical of benzophenone tends to avoid solvent mole- cules and nucleophiles, cross-linking preferentially at unreactive carbon centers. Because the photoexcitation of benzophenones is reversible, a given molecule of the analogue which is not proximal to the ligand binding site can undergo several excitation–relaxation cycles without suffering nonspecific side reactions. Thus, 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(01)00339-0 Bioorganic & Medicinal Chemistry Letters 11 (2001) 1871–1874 *Corresponding author. Tel.: +1-215-898-3158; fax: +1-215-898- 5129; e-mail: mjoullie@sas.upenn.edu