M. Eugenio Vazquez

• Dr. Organic Chemistry
• Laboratory Head @ChemBioUSC at University of Santiago de Compostela

We report a photolabile biselectrophilic Ru(ii) complex that can be used for homo- or heterodimerization of cysteine-containing peptides. The resulting dimers can be efficiently disassembled by long-wavelength light. As proof-of-concept, we describe the preparation of homo- and heterodimeric bZIP peptides whose DNA-binding properties can be turned...

A flexible and versatile synthetic approach for the construction of water-stable DNA-binding chiral peptide helicates based on the solid phase peptide synthesis (SPPS) methodology is reported.

We report the rational design of a 20-mer basic peptide, derived from the transcriptional antitermination protein N of bacteriophage P22, equipped with a luminescent DOTA[Tb3+] macrocyclic complex and a sensitizing tryptophan antenna. Folding of this peptide into an α helical conformation, which occurs upon binding to its target boxB RNA hairpin, r...

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 meta...

Intermolecular sensitization of lanthanide ions was effectively implemented in the development of fluorescent sensors targeting cyclin A. A chelating unit has been conjugated to peptides containing a known cyclin A binding motif (CBM). Upon interaction of the modified terbium-chelating peptides with the cyclin A substrate recruitment groove, the Tb...

We describe the first systematic analysis of NAF-emitting peptides in solution and show that short peptides derived from zwitterionic single α-helices (SAHs), formed exclusively by non-aromatic lysine and glutamic acid residues, are UV-active and luminescent at near-UV wavelengths in solution (λexc = 320 nm; λem ≈ 420 nm). We also show that their e...

We have synthesized two bipyridyl-based oligocationic cyclopeptides through solid phase peptide synthetic methods (SPPS) and studied their metal-binding, redox and biological properties in the presence of copper(II) ions in water media by several techniques. Remarkably, the studies performed indicate that, while the free cyclopeptides exhibit quite...

A bisbenzamidine DNA binding agent can be easily dimerized by alkylation of its terminal amidine groups to afford an extended minor groove binder with over 20-fold enhanced DNA affinity towards extended A/T-rich sites. NMR experiments in combination with molecular dynamics simulation studies provide structural insight into the insertion of this com...

By deprotonating Btp-derived ligands, precise control over counterions in the outer-coordination sphere is achieved in both neutral and charged anisotropic Co II complexes exhibiting field-induced single-ion magnetism.

Polypeptides lacking aromatic residues can display in the aggregated state anomalous spectroscopic properties, such as UV absorption and fluorescence emission. Despite the fundamental interest and potential applications of this anomalous luminescence, our understanding of this phenomenon is still poor, and no soluble model system or general design...

Epilepsies are a common and severe neurological condition characterized by spontaneous and recurrent seizures. Although anti-seizure medications are effective for most patients, about 30% remain pharmacoresistant. Moreover, uncontrolled seizures are associated with risk factors and shortened life expectancy for individuals with refractory epilepsy....

The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactiva...

The use of copper-based artificial nucleases as potential anticancer agents has been hampered by their poor selectivity in the oxidative DNA cleavage process. An alternative strategy to solve this problem is to design systems capable of selectively damaging noncanonical DNA structures that play crucial roles in the cell cycle. We designed an oligoc...

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of the...

RAS oncoproteins are molecular switches associated with critical signaling pathways that regulate cell proliferation and differentiation. Mutations in the RAS family, mainly in the KRAS isoform, are responsible for some of the deadliest cancers, which has made this protein a major target in biomedical research. Here we demonstrate that a designed b...

Self-assembled proteins are privileged building blocks for the bottom-up organization of matter at the nanoscale. However, since most proteins are very large, they have to be produced by recombinant expression, which is less versatile and flexible than chemical synthesis. Here, we show that we can bridge the potential of proteins for nanofabricatio...

Combining coordination chemistry and peptide engineering offers extraordinary opportunities for developing novel molecular (supra)structures. Here, we demonstrate that the β-annulus motif is capable of directing the stereoselective assembly of designed peptides containing 2,2'-bipyridine ligands into parallel three-stranded chiral peptide helicates...

Although largely overlooked in peptide engineering, coordination chemistry offers a new set of interactions that opens unexplored design opportunities for developing complex molecular structures. In this context, we report new artificial peptide ligands that fold into chiral helicates in the presence of labile metal ions such as FeII and CoII. Hete...

Artificial bipyridine‐containing peptide ligands encode in their sequence the stereoselective folding into chiral three‐stranded FeII or CoII peptide helicates and as kinetically inert dinuclear CoIII complexes by in‐situ oxidation. These peptide helicates selectively label DNA replication foci in functional cells. Abstract Although largely overlo...

ConspectusDNA is the molecule responsible for the storage and transmission of the genetic information in living organisms. The expression of this information is highly regulated. In eukaryotes, it is achieved mainly at the transcription level thanks to specialized proteins called transcription factors (TFs) that recognize specific DNA sequences, th...

a> Peptide engineering has been extremely successful in creating new structures with defined properties and functions. Although generally overlooked in this context, coordination chemistry offers an additional set of interactions that opens unexplored design opportunities for developing complex molecular structures. With this in mind, we report the...

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...

div>Self-assembled proteins are privileged building blocks for the bottom-up organization of matter at the nanoscale. However, since most proteins are very large, they have to be produced by recombinant expression, which is less versatile and flexible than chemical synthesis. Here, we show that we can bridge the potential of proteins for nanofabric...

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations...

Herein we present the synthesis of two ligands containing two di(2-picolyl)amine (DPA) units linked by either a 1,1'-(pyridine-2,6-diyl)bis(3-ethylurea) (L1) or a 1,1'-(1,3-phenylene)bis(3-ethylurea) (L2) spacer. The corresponding binuclear CuII and ZnII complexes were prepared and isolated. The X-ray structures of the L1 ligand and the [Cu2L1Cl2]2...

We report the first Ru(II) coordination compounds that interact with DNA through a canonical minor groove insertion mode and with selectivity for A/T rich sites. This was made possible by integrating a bis‑benzamidine minor groove DNA-binding agent with a ruthenium(II) complex. Importantly, one of the enantiomers (Δ‑[Ru(bpy)2b4bpy]2+, Δ‑4Ru) shows...

We describe the first chemical synthesis of a functional mutant of the DNA binding domain of the oncoprotein MYC, using two alternative strategies which involve either one or two Native Chemical Ligations (NCLs). Both routes allowed the efficient synthesis of a miniprotein which is capable of heterodimerizing with MAX, and replicate the DNA binding...

We report the first Ru(II) coordination compounds that interact with DNA through a canonical minor groove insertion mode and with selectivity for A/T rich sites. This was made possible by integrating a bis‑benzamidine minor groove DNA-binding agent with a ruthenium(II) complex. Importantly, one of the enantiomers (Δ‑[Ru(bpy)₂ b4bpy ]<sup...

We propose that peptides are highly versatile platforms for the precise design of supramolecular metal architectures, and particularly, for the controlled assembly of helicates. In this context, we show that the bacteriophage T4 Fibritin foldon (T4Ff) can been engineered on its N-terminus with metal-chelating 2,2′-bipyridine units that stereoselect...

The use of light‐activated triphenylphosphonium‐based antimicrobials to tackle the emerging problem of antimicrobial resistance has been proposed in this study. Chemical modification of phenalenone and perylene with triphenylphosphonium cations enhances their photoantimicrobial activity up to 10⁷ times. Selective targeting of Gram‐positive bacteria...

Light‐mediated killing of pathogens by cationic photosensitisers is a promising antimicrobial approach that avoids the development of resistance inherent to the use of antimicrobials. In this study, we demonstrate that modification of different photosensitisers with the triphenylphosphonium cation yields derivatives with excellent photoantimicrobia...

Obtaining artificial proteins that mimic the DNA binding properties of natural transcription factors could open new ways of manipulating gene expression at will. In this context it is particularly interesting to develop simple synthetic systems. Inspired by the modularity of natural transcription factors, we have designed synthetic miniproteins tha...

A set of Ru(II) metallopeptides containing the dppz ligand has been synthesized using solid-phase peptide synthesis methods. Fluorescence titration studies show that those metallopeptides featuring an octaarginine tail display a large binding preference for DNA G-quadruplex structures over those lacking the oligocationic domain, and also that the i...

Inspired by natural transcription factors (TFs), researchers have explored the potential of artificial peptides for the recognition of specific DNA sequences, developing increasingly sophisticated systems that not only display excellent DNA binding properties, but also are endowed with new properties not found in their natural counterparts. Here we...

A fragment of the DNA basic region (br) of the GCN4 bZIP transcription factor has been modified to include two His residues at designed i, and i+4 positions of its N-terminus. The resulting monomeric peptide (brHis2) does not bind to its consensus target DNA site (5’-GTCAT-3’). However, addition of cis-Pd(en)Cl2 promotes a high-affinity interaction...

We describe a lanthanide biosensor that responds to CDK4 kinase activity in melanoma cell extracts through substantial and dose-dependent increase in luminescence, thanks to sensitization of a DOTA[Tb3+] complex incorporated into a CDK4 substrate peptide by a unique tryptophan residue in an adjacent phosphoaminoacid binding moeity.

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between v...

The cell internalization of designed oligoarginine peptides equipped with six glutamic acid residues and an anionic pyranine at the N-terminus is triggered upon addition of a supramolecular host. This host binds specifically to the pyranine moiety, enabling the complex to traverse the cell membrane. Interestingly, none of the components, neither th...

Supplementary

Guanine quadruplexes (GQs) are compact four-stranded DNA structures that play a key role in the control of a variety of biological processes, including gene transcription. Bulky ruthenium complexes featuring a bipyridine, a terpyridine, and one exchangeable ligand ([Ru(terpy)(bpy)X](n+) ) are able to metalate exposed guanines present in the GQ of t...

Guanine quadruplexes (GQs) are compact four-stranded DNA structures that play a key role in the control of a variety of biological processes, including gene transcription. Bulky ruthenium complexes featuring a bipyridine, a terpyridine, and one exchangeable ligand ([Ru(terpy)(bpy)X]n+) are able to metalate exposed guanines present in the GQ of the...

Blood-based biomarkers (liquid biopsy) offer extremely valua-ble tools for the non-invasive diagnosis and monitoring of tumors. The protein c-MYC, a transcription factor that has been shown to be deregulated in up to 70% of human cancers, can be used as a robust proteomic signature for cancer. Herein, we developed a rapid, highly specific and sensi...

A series of oligoarginine peptide derivatives containing cyclometallated iridium(iii) units display remarkable cytotoxicity, comparable to that of cisplatin. In vitro studies with unilamellar vesicles support a membrane-disrupting mechanism of action.

We describe the synthesis of designed peptidic modules that self-assemble in specific DNA sequences of 12 base pairs in the presence of Ni(II) salts. The modules consist of modified fragments of transcription factors that have been appropriately engineered to include metal-chelating His and bipyridine ligands.

We report the rational design of a DNA-binding peptide construct composed of the DNA-contacting regions of two transcription factors (GCN4 and GAGA) linked through an AT-hook DNA anchor. The resulting chimera, which represents a new, non-natural DNA binding motif, binds with high affinity and selectivity to a long composite sequence of 13 base pair...

Rational modification of a natural RNA-binding peptide with a lanthanide EDTA chelator, and a phenanthroline ligand yields a highly selective luminescent sensor. The sensing mechanism relies on the RNA-triggered folding of the peptide into a β-hairpin, which promotes the coordination of the phenanthroline sensitizer, and the efficient sensitization...

We demonstrate the application of solid-phase peptide synthesis methods for assembling polynuclear Ir(III) organo-metallopeptides that exhibit high DNA-binding affinity, sequence selectivity, and high cytotoxic effect towards a set of cancer cell lines.

We have synthesized oligoarginine conjugates of selected DNA-binding agents (a bisbenzamidine, acridine, and thiazole orange), and demonstrated that their DNA binding and cell internalization can be inhibited by appending a negatively charged oligoglutamic tail through a photolabile linker. Irradiation with UV light releases the parent octaarginine...

ARC repressor (apoptosis repressor with caspase recruitment domain) is a protein which binds selectively to a specific sequence of DNA. In humans, ARC is primarily expressed in striated muscle tissue, which normally does not undergo rapid cell turnover. This suggests that ARC may play a protective role in the prevention against Duchenne Muscular Dy...

We(have(applied(solidBphase(synthesis(methods(for(the(construction(of(tris(bipyridyl)(peptidic(ligands(that(coordinate(Fe(II)( ions(with(high(affinity(and(fold(into(stable(mononuclear(metallopeptides.(The(main(factors(influencing(the(folding(pathway( and(chiral(control(of(the(peptidic(ligands(around(the(metal(ions(has(been(studied(both(by(experimen...

A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the C...

We report the development of chimeric DNA binding peptides comprised of the DNA binding fragment of natural transcription factors (basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif. The resulting peptide conjugates display high DNA affinity and excellent sequence selectivity. Furthermore, the the AT-Hook...

Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction (NMJ) of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis simultaneously occurring during nerve stimulation. We observe two endocytosis components that occur sequential...

We describe a ds-oligonucleotide-peptide conjugate that is able to efficiently dismount preformed DNA complexes of the bZIP regions of oncoproteins c-Fos and c-Jun (AP-1), and therefore might be useful as disrupters of AP-1-mediated gene expression pathways.

Conjugation of a short peptide fragment from a bZIP protein to an oligoguanidinium tail results in a DNA-binding miniprotein that selectively interacts with composite sequences containing the peptide-binding site next to an A/T-rich tract. In addition to stabilizing the complex with the target DNA, the oligoguanidinium unit also endows the conjugat...

We 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...

In recent decades there has been great interest in the design of highly sensitive sequence-specific DNA binders. The eligibility of the binder depends on the magnitude of the fluorescence increase upon binding, related to its photophysics, and on its affinity and specificity, which is, in turn, determined by the dynamics of the binding process. The...

We report the discovery of a fluorogenic dye, N1,N3-di(2-aminidonaphthalen-6-yl) propane-1,3-diamine, MitoBlue, which selectively stains functional mitochondria while displaying low toxicity, bright blue emission, and high resistance to photobleaching. Additionally, we show that a biotin-labeled MitoBlue derivative can be used as a handle for the d...

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...

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...

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 b...

Attachment of alloc protecting groups to the amidine units of fluorogenic DNA-binding bisbenzamidines or to the amino groups of ethidium bromide leads to a significant reduction of their DNA affinity. More importantly, the active DNA-binding species can be readily regenerated by treatment with ruthenium catalysts in aqueous conditions, even in cell...

Signaling 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...

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 fol...

A new bipyridine building block has been used for the solid-phase synthesis of dinuclear DNA-binding ruthenium(II) metallopeptides. Detailed spectroscopic studies suggest that these compounds bind to the DNA by insertion into the DNA minor groove. Moreover, the potential of the solid-phase peptide synthesis approach is demonstrated by the straightf...

Transcription 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 pro...

A SERS (surface-enhanced Raman scattering) based sensor was developed for the detection of the oncoprotein c-Jun at nanomolar levels. c-Jun is a member of the bZIP (basic zip-per) family of dimeric transcriptional activators and its overexpression has been associated to carcinogenic mecha-nisms in several human cancers. For our sensing purpose, we...

The synthesis of peptides with a terpyridine chelating ligand attached to a lysine side chain is described. The main advantage of this methodology relies on the introduction of the terpyridine unit while the peptide is still in the solid phase, thus avoiding reactions in solution and laborious HPLC purifications of synthetic intermediates.

One of the strategies used by nature to regulate gene expression relies on the stimuli-controlled combination of DNA-binding proteins. This in turn determines the target-binding site within the genome, and thereby whether a particular gene is activated or repressed. Here we demonstrate how a designed basic region leucine zipper-based peptide can be...

Appending negatively charged Glu(8) tails to a peptide dimer derived from the GCN4 transcription factor leads to an effective suppression of its DNA binding. The specific DNA recognition can be restored by irradiation with UV light by using a photolabile linker between the acidic tail and the DNA binding peptide.

Getting in the groove: Fluorescence correlation spectroscopy reveals that the dynamics of the association process of the bisbenzamidine minor-groove binder BBA-OG (blue with green star, see scheme) to dsDNA is not controlled by diffusion, but by the insertion of the binder into the groove at the specific site (red), as shown by the rate constants f...

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...

Ligand effects The chirality of a metallopeptide is determined by the chirality of a single proline amino acid (D‐ or L‐P) located between the two coordinating 2,2′‐bipyridine residues. In their Communication on page 7030 ff., M. Vázquez López, J. Jiménez‐Barbero, M. E. Vázquez et al. describe the synthesis as well as the structural elucidation of...

Chemical derivatization of nucleic stains such as ethidium bromide or DAPI with tailored, photoresponsive caging groups, allows for "on demand" spatiotemporal control of their in vivo nucleic acid binding, as well as for improving their cellular uptake. This effect was particularly noteworthy for a nitro-veratryloxycarbonyl-caged derivative of ethi...

Playing into our hands: The achiral bipyridine amino acid fluorenylmethyoxlcarbonyl 5-amino-3-oxapentanoic acid (Fmoc-O1PenBpy-OH) has been used for the solid-phase synthesis of metallopeptides. Circular dichroism, molecular modeling, and NMR spectroscopic studies show that the chirality of the resulting metal complexes in aqueous solution is deter...

Cysteine string protein-α (CSP-α) is a synaptic vesicle protein that prevents activity-dependent neurodegeneration by poorly understood mechanisms. We have studied the synaptic vesicle cycle at the motor nerve terminals of CSP-α knock-out mice expressing the synaptopHluorin transgene. Mutant nerve terminals fail to sustain prolonged release and the...

Caging of the amidinium moieties of two representative bisbenzamidine DNA binders with suitable photoresponsive protecting groups suppresses their DNA-binding, which can then be fully recovered upon irradiation with UV light that removes the caging groups, regenerating the parent compounds. Moreover, a caged aza-pentamidine (©1a) is inert against C...

The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein-DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non-natural age...

We report the design and development of a fluorescent sensor specifically designed to target cyclin A, a protein that plays a key role in the regulation of the cell cycle. Computational studies provide a molecular picture that explains the observed emission increase, suggesting that the 4-DMAP fluorophore in the peptide is protected from the bulk s...

Designed terbium-chelating peptides were used to monitor the formation of leucine zipper associations. The sensing strategy is based on the modulation of the antenna effect from a Trp residue donor and the luminescence increase arising from the folding that takes place upon the formation of the coiled-coil structure. An application in the specific...