Extending the Excitation Wavelength of Potential Photosensitizers via Appendage of a Kinetically Stable Terbium(III) Macrocyclic Complex for Applications in Photodynamic Therapy.

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.

Installation of a Rigid EDTA-Like Motif into a Protein α-Helix for Paramagnetic NMR Spectroscopy with Cobalt(II) Ions.

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.

DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.

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.

A facile, click chemistry-based approach to assembling fluorescent chemosensors for protein tyrosine kinases.

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.

New macrocyclic terbium(III) complex for use in RNA footprinting experiments.

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.

Lanthanide complexes as luminogenic probes to measure sulfide levels in industrial samples.

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 âˆ¼ 250 nM), while complex 1 was capable of monitoring µM levels of sulfide in partially refined crude oil.

Biomolecule detection in porous silicon based microcavities via europium luminescence enhancement.

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.

Engineering of a bis-chelator motif into a protein α-helix for rigid lanthanide binding and paramagnetic NMR spectroscopy.

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.