The development of viable photodynamic therapy protocols is often hindered by photosensitizers that require high-energy UV irradiation that has limited potential for clinical use due to its low tissue penetration. Herein, we report a strategy for extending the excitation wavelength of potential photosensitizers via the covalent attachment of a terbium(III)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate complex (DO3A-Tb). The method was systematically demonstrated with a series of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene, anthracene, pyrene, and fluoranthene) to prepare six new complexes (Tb1-Tb6) with bathochromic shifts that extended into the visible region. Determination of their quantum yields for singlet oxygen (1O2) production at 350 and 420 nm showed significant enhancements from the parent molecule in all cases. Cell viability studies on cervical cancer cells (HeLa) and noncancerous MRC-5 cells showed no measurable cytotoxicity for all complexes prior to light irradiation. However, after irradiation at 420 nm (20 min, 9.27 J cm-2), Tb3-Tb6 were phototoxic to HeLa cells with IC50 values between 14.3-32.3 μM. Cell morphological studies and fluorescence microscopy with live/dead cell stains confirmed these findings. In addition, these complexes were highly stable in human blood plasma, with no significant degradation observed after 96 h at 37 °C. This excellent phototoxicity profile and high stability in blood plasma, coupled with the moderately lipophilic nature of the complexes, favorably indicate the potential of DO3A-Tb as a heavy atom-bearing moiety for modification of potential photosensitizers into ideal phototherapeutic drug candidates with longer excitation wavelengths for in vivo application.

Coupling two copies of an iminodiacetic acid-cysteine hybrid ligand to a pair of cysteine residues positioned in an i, i+4 arrangement within a protein α-helix leads to generation of an EDTA-like metal ion-binding motif. Rigid binding of a Co(II) ion by this motif produces pseudo-contact shifts suitable for paramagnetic NMR structural studies.

A series of lanthanide-based, azide-appended complexes were investigated as hydrogen sulfide-sensitive probes. Europium complex 1 and Tb complex 3 both displayed a sulfide-dependent increase in luminescence, while Tb complex 2 displayed a decrease in luminescence upon exposure to NaHS. The utility of the complexes for monitoring sulfide levels in industrial oil and water samples was investigated. Complex 3 provided a sensitive measure of sulfide levels in petrochemical water samples (detection limit ∼ 250 nM), while complex 1 was capable of monitoring μM levels of sulfide in partially refined crude oil.

In this paper, we demonstrate the detection of europium-complex-labeled streptavidin in a porous silicon microcavity (pSiMC) via luminescence enhancement. The pSiMC platform was modified for optimized luminescence enhancement which encompassed changing the pore size of the microcavity to ensure molecular infiltration and adjusting the optical quality of the microcavity. Characterization of the optimized surface was performed by infrared spectroscopy, interferometric reflectance spectroscopy and luminescence measurements. Luminescence enhancement of the bound Eu(III) complex by a factor of 3 was observed on the optimized pSiMC as compared to that on a single pSi layer. The ability of a pSiMC to act as a luminescence enhancing sensor was confirmed using streptavidin as a model analyte on a biotin-modified pSiMC. The sensor was able to detect Eu(III) complex labeled streptavidin with a concentration as low as 150 nM. Furthermore, streptavidin was selectively detected when spiked in human wound fluid. The concept of detecting Eu(III) labeled bioconjugates on pSiMC may be incorporated into the design of highly sensitive and specific point-of-care biosensors.

A group of fluorophore-labeled peptide substrates of Src kinases have been synthesized with the aid of click chemistry. Some of the generated peptides exhibit an increase in fluorescence upon phosphorylation and are capable of detecting Src kinases with high sensitivity and specificity. Their availability permits real-time activity measurement of aberrantly activated oncogenic Src kinases in the crude lysate of chronic myelogenous leukemia cells. These new chemosensor peptides are highly useful tools that can be used for high-throughput screening to search for small molecule inhibitors of Src kinases as potential therapeutics for cancer treatment.

Attachment of two nitrilotriacetic acid-based ligands to a protein α-helix in an i, i + 4 configuration produces an octadentate chelating motif that is able to bind paramagnetic lanthanide ions rigidly and with high affinity, leading to large pseudocontact shifts and residual dipolar couplings in the NMR spectrum.

All the way with IDA! Attachment of iminodiacetic acid (IDA) to a protein helix creates a rigid lanthanide binding site that can be exploited for paramagnetic NMR spectroscopy (see picture). Pseudo-contact shifts (PCSs) larger than 8 ppm are achievable with the tag, and metal exchange is sufficiently fast to enable signal assignment by 15Nz exchange spectroscopy, eliminating the need for an initial protein model. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.

ADVERTISEMENT RETURN TO ISSUEPerspectiveNEXTTripeptide Motifs in Biology: Targets for Peptidomimetic DesignPhuc Ung†‡ and David A. Winkler*†‡View Author Information† CSIRO Materials Science and Engineering, Bag 10, Clayton South MDC 3169, Australia‡ Monash Institute of Pharmaceutical Science, Parkville 3152, Australia*Phone: +61 3 9545 2477. Fax: +61 3 9545 2446. E-mail: [email protected]Cite this: J. Med. Chem. 2011, 54, 5, 1111–1125Publication Date (Web):January 28, 2011Publication History Received8 October 2010Published online28 January 2011Published inissue 10 March 2011https://doi.org/10.1021/jm1012984Copyright © 2011 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views3798Altmetric-Citations54LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (2 MB) Get e-AlertscloseSUBJECTS:Antagonists,Cells,Peptides and proteins,Receptors,Small molecules Get e-Alerts

A group of fluorescence-based chemosensor peptide substrates of Src kinases were synthesized via the click chemistry approach. Some of the generated chemosensor peptides are capable of detecting Src kinases with high sensitivity and specificity. Their availability permits real-time activity measurement of the aberrantly activated oncogenic Src kinases in crude lysate of chronic myelogenous leukaemia cells. Furthermore, these chemosensor peptides are amenable for high throughput screens to search for small molecule inhibitors of Src kinases as potential therapeutics for cancer treatment Aberrant activation and expression of Src-family of protein tyrosine kinases (SFKs) are known to contribute to the formation and disease progression of many types of cancer such as leukaemia, colon and breast carcinoma. For example, SFKs cooperate with the oncogenic tyrosine kinase Bcr-Abl to induce oncogenic effects in chronic (1) Hoshino, K.; Quintas-Cardama, A.; Yang, H.; SanchezGonzalez, B.; Garcia-Manero, G. Leukemia 2007, 21, 906. (2) Scheijen, B.; Griffin, J. D. Oncogene 2002, 21, 3314. myelogenous leukaemia (CML). For this reasons, small molecule compounds capable of specific inhibition of SFKs are potential therapeutics for cancer treatment. To facilitate the search and development of these therapeutic small molecule SFK inhibitors, we have designed and synthesized fluorescent chemosensor peptides suitable for high throughput assay for activitiy of SFKs. (3) Donato, N. J.; Wu, J. Y.; Stapley, J.; Gallick, G.; Lin, H.; Arlinghaus, R.; Talpaz, M. Blood 2003, 101, 690 Herein, we describe how we used the click chemistry approach in synthesis and assembly of these fluorescent chemosensor peptides. The approach we used is suitable for rapid syntheses and screning of a small library of peptide substrates derived with different types of fluorophores for use as chemosensor substrates of protein kinases. Furthermore, we used one of the peptide substrates chemosensors and examined its suitability for rapid and convenient assay of SFK activity in crude cell lysate of CML cells with and without treatment with CML therapeutic compound Imatinib. The Src optimal peptide with the sequence of AEEEIYEGEFEA was previously found to be a selective and efficient peptide substrates of SFKs. The phenylalanine at the pY+3 position was determined to be an essential determinants dictating efficient phosphorylation of the Src optimal peptide by SFKs. Replacement of phenylalanine by alanine significantly reduced the efficiency of phosphorylation of Src optimal peptide by SFKs, indicating that SFKs possess a specific binding pocket for a hydrophobic residue at the pY + 3 position of the protein and peptide substrates. Based upon these findings, we proposed to introduce a number of fluorophores at the pY+3 position of a peptide scafold derived from the Src-optimal peptide. Since the fluorophore moiety of the resultant peptide analogs can potentially bind to the (pY+3)-binding pocket of SFKs, these peptides are expected to be efficient and specific substrates of SFKs. One of the criteria for a Src-optmal peptide analog to serve as a chemosensor peptide of SFKs is its ability to exhibit fluorescence enhancement upon phosphorylation. Lawrence et al. (REF) previously developed a pyrene-based chemosensor peptide substrate of which the phenol group of the tyrosine quenches the fluorescence of the pyrene-moiety. Upon phosphorylation, formation of phosphoester in the resultant phosphotyrosine disrupts the dynamic interaction between the aromatic groups of the phosphotyrosine and the pyrene moiety. In this manuscript, we describe convenient methods to synthesize a library of Src-optimal peptide analogs. We also present results of the examination of the efficiency and specificity of phosphorylation of these peptides by SFKs as well as the extent of fluorescence enhancement of these peptides induced by their phosphorylation by SFKs. The peptide scaffold was prepared by replacing the hydrophobic phenylalanine at the pY + 3 position of the Src optimal peptide with propargylglycine. The peptide (referred to as propargylglycince – SOP) contains the sequence AEEEIYGE(Pra)EAKKKK-NH2 in which the – NH2 represents the amidated C terminus and Pra represents the propargylglycine residue. The reactive alkyne group of the propargylglycine residue allows it to react with a large number of azide derivatized fluorophores to generate multiple Src optimal peptide analogues carrying a wide spectrum of fluorophores at the pY + 3 position. The approach allows the peptide scaffold (4) Songyang, Z.; Carraway, K. L., 3rd; Eck, M. J.; Harrison, S. C.; Feldman, R. A.; Mohammadi, M.; Schlessinger, J.; Hubbard, S. R.; Smith, D. P.; Eng, C.; et al. Nature 1995, 373, 536. (5) Sicilia, R. J.; Hibbs, M. L.; Bello, P. A.; Bjorge, J. D.; Fujita, D. J.; Stanley, I. J.; Dunn, A. R.; Cheng, H. C. J Biol Chem 1998, 273, 16756. (6) Waters, M. L. Curr Opin Chem Biol 2002, 6, 736. (7) Hunter, C. A.; Lawson, K. R.; Perkins, J.; Urch, C. J. The Royal Society of Chemistry 2001, 2, 651. (8) Wang, Q.; Cahill, S. M.; Blumenstein, M.; Lawrence, D. S. J Am Chem Soc 2006, 128, 1808. to “click’’ with a library of azide – derivatized fluorophores as refered as the click chemistry approach. The general scheme for the synthesis of the fluorescent Src optimal peptide analogues is depicted in Scheme 1. A-E-E-E-I-Y-G-E-G-E-A-K-K-K-K-NH2

Reaction of terbium triflate with a heptadentate ligand derivative of cyclen, L1 = 2-[7-ethyl-4,10-bis(isopropylcarbamoylmethyl)-1,4,7,10-tetraazacyclododec-1-yl]-N-isopropyl-acetamide, produced a new synthetic ribonuclease, [Tb(L1)(OTf)(OH(2))](OTf)(2).MeCN (C1). X-ray crystal structure analysis indicates that the terbium(III) center in C1 is 9-coordinate, with a capped square-antiprism geometry. While the terbium(III) center is tightly bound by the L1 ligand, two of the coordination sites are occupied by labile water and triflate ligands. In water, the triflate ligand is likely to be displaced, forming [Tb(L1)(OH(2))(2)](3+), which is able to effectively promote RNA cleavage. This complex greatly accelerates the rate of intramolecular transesterification of an activated model RNA phosphodiester, uridine-3'-p-nitrophenylphosphate (UpNP), with k(obs) = 5.5(1) x 10(-2) s(-1) at 21 degrees C and pH 7.5, corresponding to an apparent second-order rate constant of 277(5) M(-1) s(-1). By contrast, the analogous complex of an octadentate derivative of cyclen featuring only a single labile coordination site, [Tb(L2)(OH(2))](OTf)(3) (C2), where L2 = 2-[4,7,10-tris(isopropylcarbamoylmethyl)-1,4,7,10-tetraazacyclododec-1-yl]-N-isopropyl-acetamide, is inactive. [Tb(L1)(OH(2))(2)](3+) is also capable of hydrolyzing short transcripts of the HIV-1 transactivation response (TAR) element, HIV-1 dimerization initiation site (DIS) and ribosomal A-site, as well as formyl methionine tRNA (tRNA(fMet)), albeit at a considerably slower rate than UpNP transesterification (k(obs) = 2.78(8) x 10(-5) s(-1) for TAR cleavage at 37 degrees C, pH 6.5, corresponding to an apparent second-order rate constant of 0.56(2) M(-1)s(-1)). Cleavage is concentrated at the single-stranded "bulge" regions of these RNA motifs. Exploiting this selectivity, [Tb(L1)(OH(2))(2)](3+) was successfully employed in footprinting experiments, in which binding of the Tat peptide and neomycin B to the bulge region of the TAR stem-loop was confirmed.