Peptide tertiary structure nucleation by side-chain crosslinking with metal complexation and double "click" cycloaddition.


Stabilized helical peptides show potential as therapeutics capable of modulating protein function through enhanced helix–protein binding. Helix-turn stabilization may be accomplished though covalent side-chain crosslinking on one face of the helix, in the i and i+4 or i+7 positions, or replacement of a main-chain hydrogen bond with a covalent linker. 5,6] We report herein a new helix-nucleating i and i+4 crosslinking strategy based on coppercatalyzed azide–alkyne [3+2] “click” cycloaddition, and demonstrate the ability of this method and metal complexation to restore coiled-coil dimerization in a folding-incompetent sequence crippled by two helix-breaking glycine residues. Elegant applications of azide–alkyne cycloaddition chemistry in peptide conjugation and structure stabilization are known in the literature; this design strategy complements known intramolecular methods for structure nucleation, such as ringclosing metathesis (RCM), and allows convergent installation of functional groups pendant to the bis-alkyne linker that could be used to modulate peptide binding, targeting, and membrane permeability. We studied 21-residue sequences derived from the GCN4 leucine zipper in which the central heptad contains glycine residues in the c and e helix positions and crosslinking residues (X) in the b and f (i and i+4) positions, leaving the hydrophobic core residues in the a and d positions intact as isoleucine and leucine, respectively. Metal complexation with i and i+4 X=His residues is known to induce monomeric helix folding, though it has not been previously demonstrated to restore structure in peptides containing two glycine residues. We postulated that the bis-triazole product of a double [3+2] cycloaddition between i and i+4 azidoalanine (Az) residues and a bis-alkyne could yield a nonlabile covalent linker isosteric with i and i+4 His–His metal complex (Figure 1A). We preFigure 1. A) Strategies for helix structure nucleation with i and i+4 crosslinking by using metal complexation (top) or double-click cycloaddition (bottom). a) Diazidoalanine peptide was treated in aqueous buffer with bis alkynes 6–9, sodium ascorbate, CuSO4, and bathophenanthroline disulfonic acid or on resin in DMF with CuI and DIEA. B) Peptide sequences 1–5 used in this study, with helical wheel positions shown above, here X=Az or His and ABA=acetamidobenzoate (e270=18000m 1 cm ). Dimerization-inhibiting mutations in 4 and 5 are underlined. C) Bis-alkyne linkers 6–9. D) Properties of stabilized and unstabilized peptides.

DOI: 10.1002/cbic.200800040

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@article{Torres2008PeptideTS, title={Peptide tertiary structure nucleation by side-chain crosslinking with metal complexation and double "click" cycloaddition.}, author={Oscar Torres and Deniz Y{\"{u}ksel and Matt Bernardina and Krishna Kumar and Dennis Bong}, journal={Chembiochem : a European journal of chemical biology}, year={2008}, volume={9 11}, pages={1701-5} }