2258 | Chem. Commun., 2014, 50, 2258--2260 This journal is © The Royal Society of Chemistry 2014 Cite this: Chem. Commun., 2014, 50, 2258 The bba fold of zinc finger proteins as a ‘‘natural’’ protecting group. Chemoselective synthesis of a DNA-binding zinc finger derivative† Je ´ ssica Rodrı ´ guez, Jesu ´ s Mosquera, Olalla Va ´ zquez, M. Eugenio Va ´ zquez* and Jose ´ L. Mascaren ˜ as* We report the selective modification of cysteine residues engineered in peptides that have two additional cysteine residues as part of a Cys 2 His 2 zinc finger motif. The chemoselective modification is achieved, thanks to the protecting effect exerted by the zinc cation upon coordination with the native cysteines and histidines of the zinc-finger fold. The strategy allows a straightforward synthesis of DNA binding zinc finger constructs. The covalent modification of proteins is a powerful strategy to modulate their macromolecular function. 1 Nature accomplishes such alterations through a variety of post-translational modifications that modulate the properties of proteins. 2 Inspired by nature, scientists have long pursued the development of methodologies that could emulate these natural post-conjugation reactions and allow the introduction of desired groups or labels in specific sites of proteins. 1,3 For a reaction to be of general use, it should selectively target the residue of interest in the presence of competing side chains of the unprotected polypeptide, and in aqueous environments. Many of the strategies developed so far for the site-selective modification of proteins rely on the introduction of designed amino acids equipped with orthogonally reactive groups that can be coupled to external reactants, for instance, by using the well known click chemistry. 4 Although this strategy allows high levels of selectivity, it requires the introduction of non-natural amino acids. 5 Alternatively, one could pursue the modification of specific natural amino acids in proteins, but achieving a good combination of reactivity and chemoselectivity is extremely challenging. In this context, most of the successful modifications of unprotected pep- tides or proteins have relied on the nucleophilic reactivity window provided by the sulfur atom in cysteine side chains, 6 which allowed a number of selective alkylations with a variety of electrophiles. A major problem of this strategy arises when the peptides or proteins of interest have more than one cysteine residue, because of the competitive formation of polyalkylated products. 7 Here, we report the selective modification of cysteine residues incorporated into a zinc finger peptide that has two additional cysteines as part of a classical Cys 2 His 2 motif. In the presence of Zn(II) these cysteines are trapped in the bba secondary fold and thereby exhibit a much decreased reactivity (Fig. 1). The ligation provides a straightforward approach to bisbenzamidine–zinc finger conjugates capable of binding to designed DNA target sequences. Since the zinc finger is a natural motif present in many transcription factors, this work opens a door for the chemoselective access to zinc finger derivatives, and for the modification and/or tagging of zinc finger proteins. The zinc finger family is the largest among eukaryotic transcrip- tion factors, and is responsible for regulating the expression of a myriad of genes controlling fundamental cellular programs. 8 The DNA recognition by zinc finger proteins has served as inspiration for the engineering of a large variety of highly appealing artificial gene regulators. 9 Despite the relevance of zinc finger proteins, work towards their selective chemical modification has been very scarce. There are a number of reports on the reactivity of zinc thiolates of different zinc fingers that suggest that, while the cysteines in charged Zn(Cys) 4 and Zn(Cys) 3 His motifs react with electrophiles or oxidants, neutral Zn(Cys) 2 (His) 2 are less reactive. 10 Therefore, we reasoned that zinc finger structures might provide a natural protec- tion for the two cysteines involved in the coordination to the metal Fig. 1 Schematic illustration of the approach for the chemoselective synthesis of zinc finger derivatives. (a) Unfolded peptide with three nucleophilic sites; (b) zinc-promoted folding and cysteine coordination leaves only a single nucleophilic site for modification. Centro Singular de Investigacio´n en Quı ´mica Biolo´xica e Materiais Moleculares (CIQUS), Departamento de Quı ´mica Orga ´nica. Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain. E-mail: joseluis.mascarenas@usc.es; Fax: +34 981 595 012; Tel: +34 981 576 541-14405 † Electronic supplementary information (ESI) available: Peptide synthesis, full experimental procedures and analytical data of the peptides and products obtained. See DOI: 10.1039/c3cc47599a Received 3rd October 2013, Accepted 19th December 2013 DOI: 10.1039/c3cc47599a www.rsc.org/chemcomm ChemComm COMMUNICATION Published on 20 December 2013. Downloaded on 07/02/2014 12:28:24. View Article Online View Journal | View Issue