Advances in lanthanide‐based luminescent peptide probes for monitoring the activity of kinase and phosphatase

This is the peer reviewed version of the following article: Pazos, E. and Vázquez, M. E. (2014), Advances in lanthanide‐based luminescent peptide probes for monitoring the activity of kinase and phosphatase. Biotechnology Journal, 9: 241-252. doi:10.1002/biot.201300203, which has been published in final form at https://doi.org/10.1002/biot.201300203. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsSignaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide‐based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide‐based luminescent peptide sensorsWe thank the support of the Spanish government’s grants SAF2010-20822-C02, CTQ2012-31341, CTQ2009-14431,SAF2007-61015 and the Xunta de Galicia INCITE09 209 084PR, GRC2010/12, PGIDIT08CSA-047209PR. E.P.thanks the Fundación Barrié de la Maza for her postdoc-toral fellowshipS

Sequence‐Selective DNA Recognition with Peptide–Bisbenzamidine Conjugates

This is the peer reviewed version of the following article: Sánchez, M. I., Vázquez, O. , Vázquez, M. E. and Mascareñas, J. L. (2013), Sequence‐Selective DNA Recognition with Peptide–Bisbenzamidine Conjugates. Chem. Eur. J., 19: 9923-9929, which has been published in final form at https://doi.org/10.1002/chem.201300519. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsTranscription factors (TFs) are specialized proteins that play a key role in the regulation of genetic expression. Their mechanism of action involves the interaction with specific DNA sequences, which usually takes place through specialized domains of the protein. However, achieving an efficient binding usually requires the presence of the full protein. This is the case for bZIP and zinc finger TF families, which cannot interact with their target sites when the DNA binding fragments are presented as isolated monomers. Herein it is demonstrated that the DNA binding of these monomeric peptides can be restored when conjugated to aza‐bisbenzamidines, which are readily accessible molecules that interact with A/T‐rich sites by insertion into their minor groove. Importantly, the fluorogenic properties of the aza‐benzamidine unit provide details of the DNA interaction that are eluded in electrophoresis mobility shift assays (EMSA). The hybrids based on the GCN4 bZIP protein preferentially bind to composite sequences containing tandem bisbenzamidine–GCN4 binding sites (TCAT⋅AAATT). Fluorescence reverse titrations show an interesting multiphasic profile consistent with the formation of competitive nonspecific complexes at low DNA/peptide ratios. On the other hand, the conjugate with the DNA binding domain of the zinc finger protein GAGA binds with high affinity (KD≈12 nM) and specificity to a composite AATTT⋅GAGA sequence containing both the bisbenzamidine and the TF consensus binding sitesWe thank the financial support provided by the Spanish grants Consolider Ingenio 2010 (SAF2010‐20822‐C02, CTQ2009‐14431/BQU, CTQ2012‐31341, CSD2007‐00006), the Xunta de Galicia (GRC2010/12, INCITE09 209 084PR, PGIDIT08CSA‐047209PR). M.I.S. thanks the Ministerio de Educación, Cultura y Deporte for his PhD fellowshipS

The ββα fold of zinc finger proteins as a “natural” protecting group. Chemoselective synthesis of a DNA-binding zinc finger derivative

We report the selective modification of cysteine residues engineered in peptides that have two additional cysteine residues as part of a Cys2His2 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 constructsWe acknowledge the support from the Spanish grants SAF2010-20822-C02, CTQ2009-14431/BQU, CSD2007-00006, the Xunta de Galicia GRC2010/12, GRC2013-041 and INCITE09 209 084PR. J. R. thanks the Fundación Gil Dávila and the Xunta de Galicia and J. M. the Spanish MCINN for their PhD fellowshipsS

Detection of phosphorylation states by intermolecular sensitization of lanthanide–peptide conjugates

The luminescence of a designed peptide equipped with a coordinatively-unsaturated lanthanide complex is modulated by the phosphorylation state of a serine residue in the sequence. While the phosphorylated state is weakly emissive, even in the presence of an external antenna, removal of the phosphate allows coordination of the sensitizer to the metal, yielding a highly emissive supramolecular complexWe thank the financial support provided by the Spanish grants SAF2010-20822-C02, CTQ2009-14431/BQU, CSD2007-00006, Consolider Ingenio 2010, the Xunta de Galicia GRC2010/12, INCITE09 209 084PR, PGIDIT08CSA-047209PR, Slovenian Research Agency (grant P1-170 to M.G.). E. P. thanks the Xunta de Galicia for her postdoctoral contractS

Selective DNA-Binding by Designed Bisbenzamidine-Homeodomain Chimeras

We report the construction of conjugates between three variants of the helix 3 region of a Q50K engrailed homeodomain and bisbenzamidine minor-groove DNA binders. The hybrid featuring the sequence of the native protein failed to bind to DNA; however, modifications that increased the α-helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interactionMinisterio de Economía y Competitividad. Grant Numbers: SAF2010-20822-C02, CTQ2012-31341 Consolider Ingenio. Grant Number: 2010 CSD2007-00006 Xunta de Galicia. Grant Number: GRC2013/14 European Regional Development Fund European Research Council. Grant Number: Na 340055 Ministerio de Ciencia e Innovación Fundación Gil DávilaS

Straightforward access to bisbenzamidine DNA binders and their use as versatile adaptors for DNA-promoted processes

Bisbenzamidines are an important family of minor groove DNA-binding agents. We present a one-step synthesis of aromatic aza-bisbenzamidines that allows straightforward and versatile access to a large number of these molecules. One of them, the azide-aza-bisbenzamidine 13, can be readily modified via click-chemistry with a variety of functionalities that can, therefore, be delivered to the vicinity of an A/T-rich DNA minor groove. This strategy, therefore, provides a simple means for triggering site selective, DNA-promoted biochemical and physicochemical processesS

Assembly of a Ternary Metallopeptide Complex at Specific DNA Sites Mediated by an AT‐Hook Adaptor

The nickel(II)‐mediated self‐assembly of a multimeric DNA binder is described. The binder is composed of two metal‐chelating peptides derived from a bZIP transcription factor (brHis2) and one short AT‐hook domain equipped with two bipyridine ligands (HkBpy2). These peptides reversibly assemble in the presence of NiII ions at selected DNA sequences of 13 base pairsFinancial support from the Spanish grants SAF2016‐76689‐R, RTI2018‐099877‐B‐I00, Orfeo‐cinqa network CTQ2016‐81797‐REDC, the Xunta de Galicia (2015‐CP082, ED431C‐2017/19 and Centro Singular de Investigación de Galicia accreditation 019–2022, ED431G 2019/03), the European Union (European Regional Development Fund—ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. S.L.‐A. thanks the Spanish MINECO for her FPI fellowship (BES‐2017‐080555); J.R. thanks the Xunta de Galicia for her PhD fellowshipS

Nickel-promoted recognition of long DNA sites by designed peptide derivatives

NOTICE: This is the peer reviewed version of the following article: Jessica Rodríguez, Jesús Mosquera, M. Eugenio Vázquez*, José L. Mascareñas* (2016), Nickel-promoted Recognition of long DNA sites by designed Peptide Derivatives. ChemEurJ., 22 (38), 13474-13477 [doi:10.1002/chem.201602783]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for selfarchivingWe describe the synthesis of designed peptidic modules that self-assemble in specific DNA sequences of 12 base pairs in the presence of NiII salts. The modules consist of modified fragments of transcription factors that have been appropriately engineered to include metal-chelating His and bipyridine ligandsWe are thankful for the support given by the Spanish grants SAF2013-41943-R, CTQ2015-70698-R, and CTQ2013-49317-EXP, the Xunta de Galicia GRC2013-041, the ERDF, and the European Research Council (Advanced Grant No. 340055). Support of COST Action CM1105, COST CM1306 and the orfeo-cinqa network are also kindly acknowledged. J.R. thanks the Xunta de Galicia for a PhD fellowshipS

Synthetic peptides caged on histidine residues with a bisbipyridyl ruthenium(II) complex that can be photolyzed by visible light

NOTICE: This is the peer reviewed version of the following article: Mosquera, J., Sánchez M. I., Mascareñas, J. L., Vázquez, M. E. (2015), Synthetic Peptides Caged on Histidine Residues with a Bisbipyridyl Ruthenium (II) Complex that Can Be Photolyzed by Visible Light. ChemComm., 51, 5501-5504[doi: 10.1039/C4CC08049A]. This article may be used for non-commercial purposes in accordance with RSC Terms and Conditions for self-archivingWe report a light-sensitive histidine building block for Fmoc/tBu solid-phase peptide synthesis in which the imidazole side chain is coordinated to a ruthenium complex. We have applied this building block for the synthesis of caged-histidine peptides that can be readily deprotected by irradiation with visible light, and demonstrated the application of this approach for the photocontrol of the activity of Ni(II)-dependent peptide nucleasesWe are thankful for the support given by the Spanish grants SAF2013-41943-R and CTQ2012-31341, the Xunta de Galicia GRC2013-041, the ERDF and the European Research Council (Advanced Grant 340055). Support of COST Action CM1105 is kindly acknowledged. J.M. and M.I.S. thank the Spanish MCINN for their PhD fellowships. Thanks also to Professor Eric C. Long at Indiana University-Purdue University Indianapolis for sharing his structural data of the RGH(Ni) complex with the DNA. We are also grateful to Prof. Wajih Al-Soufi, from the Department of Physical Chemistry at the Universidad de Santiago de Compostela, for his help in the construction of the LED photolysis apparatus

Reversible supramolecular assembly at specific DNA sites: Ni-promoted, bivalent DNA binding with designed peptide and bipyridylbisbenzamidine components

At specific DNA sites, nickel(II) salts promote the assembly of designed components, namely a bis(histidine)-modified peptide that is derived from a bZIP transcription factor and a bis(benzamidine) unit that is equipped with a bipyridine. This programmed supramolecular system with emergent properties reproduces some key characteristics of naturally occurring DNA-binding proteins, such as bivalence, selectivity, responsiveness to external agents, and reversibility.We are thankful for support from Spanish grants (SAF2010-20822-C02 and CTQ2012-31341), Consolider Ingenio 2010 (CSD2007-00006), the Xunta de Galicia (GRC2013-041, INCITE09 209084PR, and PGIDIT08CSA-047209PR), the ERDF, and the European Research Council (Advanced Grant 340055). J.M. and M.I.S. thank the Spanish MCINN for their Ph.D. fellowships.S