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Fig. 3. Comparison of conventional (A)and reverse (B) thioether ligationapproaches (triangle denotes thioetherlinkage).(Figure 3B) [2], compounded by the highexcess of thiol-functionalized epitope required,and the problem cannot be solved by TCEPin situ reduction, since exposure to highamounts of phosphine blocks reactive sites inthe ClAc-functionalized core. In contrast, inthe reverse format, TCEP amounts are lowenough to pose minimal damage to ClAcgroups of the peptide epitope. Another benefitof in situ TCEP reduction is that it allowsemploying much less peptide. Thus, a standardligation using a tetravalent ClAc core typicallyrequires at least 16 equiv of thiolfunctionalizedpeptide (4 equiv / branch), ofwhich a great deal becomes lost todimerization. In contrast, the reverse formatneeds only 2 equiv / branch of ClAc peptide.All this ensures that ligations can be efficientlydriven to quantitative replacement at all thiolsites, vs. the partial replacement levelsinevitably encountered in standard thioetherligations. As a result, the end products aremore homogeneous and easier to purify(Figures 2 and 3B).Given the well-known advantages of solidphase vs. solution chemistry, we have alsoexplored the feasibility of reverse thioetherligations in the solid phase. Among theanticipated advantages of such an approach arenot only efficiency and cleanliness, withminimal purification required, but more importantly the possibility of (1) using watercompatibleresins (e.g. ChemMatrix), and (2) driving ligations to completion by submittingthe resin-bound thiol core to repeated alkylation (ClAc epitope) and reduction (TCEP)cycles, with intermediate washing steps. The resin-bound version of the polythiol core b(Figure 1) is not achievable by Mpa acylation of a standard poly-Lys core-resin, becausecomplete on-resin removal of the S-Trt group is difficult. As an alternative, we have usedacylation with dithiodipropionic acid followed by quantitative TCEP treatment. Thusprepared, the resin-bound polythiol platform can be efficiently used in reverse thioetherligation reactions similar to the ones described above.AcknowledgementsW.K. is a fellow in the Juan de la Cierva Program of the Spanish Ministry of Science and Innovation(MICINN). Work supported by grants BIO2008-04487-CO3-02 from MICINN, and SGR2005-00494and SGR2009-00494 from Generalitat de Catalunya.References1. Tam, J.P. Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413 (1988).2. Kowalczyk, W., De la Torre, B.G., Andreu, D. Bioconj. Chem. 21, 102-110 (2010).223