New 9- or 10-dentate luminescent lanthanide chelates

Polyaminocarboxylate-based luminescent lanthanide complexes have unusual emission properties, including millisecond excited-state lifetimes and sharply spiked spectra compared to common organic fluorophores. There are three distinct sections in the structure of the luminescent lanthanide chelates: a polyaminocarboxylate backbone to bind the lanthanide ions tightly, an antenna molecule to sensitize the emission of lanthanide ions, and a reactive group to attach to biomolecules. We have previously reported the modifications on the chelates, on the antenna molecules (commonly cs124), and on the reactive sites. In searching for stronger binding chelates and better protection from solvent hydration, here we report the modification of the coordination number of the chelates. A series of 9- and 10-dentate chelates were synthesized. Among them, the 1-oxa-4,7-diazacyclononane (N2O)-containing chelate provides the best protection to the lanthanide ions from solvent molecule attack, and forms the most stable lanthanide coordination compounds. The TTHA-based chelate provides moderately good protection to the lanthanide ions.     

Thiol-reactive luminescent lanthanide chelates: part 2

Luminescent lanthanide complexes have unusual spectroscopic characteristics, including millisecond excited-state lifetime and sharply spiked emission spectra. These characteristics make them valuable alternatives to conventional organic fluorescent probes in detection applications and for measuring nanometer-scale conformational changes in biomolecules via resonance energy transfer. Our group has previously reported the syntheses and application of various luminescence complexes that have polyaminocarboxylate chelates coupled to a carbostyril antenna and thiol or amine-reactive groups. Here we report the syntheses of new thiol-reactive forms of DTPA-cs124 chelates, including two iodoacetamide forms (phenylalanine-iodoacetamide and ethylenediamine iodoacetamide) and two methane-thio-sulfonate forms (ethylmethanethiosulfonate and carboxyethylmethanethiosulfonate). In addition, we have developed an improved synthesis of a previously reported maleimide form.     

On-resin assembly of a linkerless lanthanide(III)-based luminescence label and its application to the total synthesis of site-specifically labeled mechanosensitive channels

A synthesis strategy for the on-resin assembly of luminescent lanthanide chelates from commercially available compounds was developed. Advantages of the approach include the absence of spacers between the metal ion and the attachment site, and the compatibility with typical chemical protein synthesis protection schemes. Methoxycoumarin-labeled lysine and tris(tert-butyl)-DOTA were consecutively coupled with high efficiency to a free amino group in otherwise fully protected peptide segments using standard peptide synthesis methods. Addition of stoichiometric amounts of Tb(3+) to the modified, cleaved, and purified peptides yielded the desired lanthanide chelate. Incorporation of this label into a chemically synthesized, full-length mechanosensitive channel of large conductance (MscL) of E. coli and subsequent reconstitution into vesicles resulted in a functional mechanosensitive channel of comparable conductance to the wild-type channel. However, this channel required increased suction to gate. Excitation of the antenna molecule methoxycoumarin at 336 nm resulted in an emission spectrum typical for Tb(3+) and a luminescence lifetime of 0.67 ms. The location of the probe close to the backbone of this protein may provide precise information about conformational changes during channel opening from LRET studies.     

Lanthanide(III) complexes of pyridine-tetraacetic acid-glycoconjugates: synthesis and luminescence studies of mono and divalent derivatives

A potent lanthanide chelate, fulfilling the requirements for the development of MRI contrast agents or luminescent probes, was armed with alkyne groups. We then implemented a click methodology to graft the bifunctional ligand to azide-containing glucoside and maltoside scaffolds. The resulting hydrophilic glycoconjugates retained the ligand binding capacity for Eu(3+) or Tb(3+) ion as evidenced by the number of bound water molecules to the lanthanide ion. Divalent Eu(3+) and Tb(3+) complexes were shown to double the brightness of the emitted fluorescent signal compared to its monovalent derivatives. Designing multivalent lanthanide luminescent probes would enable the fluorescent signal of labeled biomolecules to be enhanced.     

A dual lanthanide probe suitable for optical (Tb3+ luminescence) and magnetic resonance imaging (Gd3+ relaxometry)

A new polyaminocarboxylate ligand derived from N,C-pyrazolylpyridine was synthesized. The luminescence and relaxometry properties of its Tb(3+) and Gd(3+) chelates were investigated in aqueous solutions. The Tb(3+) chelate is strongly luminescent having remarkable lifetime and quantum yield (tau=1.82ms and Phi=0.42). The 1/T(1) proton relaxivity at 20MHz and 25 degrees C (5.3s(-1)mM(-1)) of the Gd(3+) chelate was found to be comparable to that of the clinically used Gd-DTPA.     

A homogeneous DNA hybridization system by using a new luminescence terbium chelate

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb(3+) (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (lambda(ex) = 548 nm and lambda(em) = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb(3+) chelate at the 3'-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb(3+) chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb(3+) chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb(3+) chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.     

Distances between tropomyosin sites across the muscle thin filament using luminescence resonance energy transfer: evidence for tropomyosin flexibility

To obtain information about the interaction of tropomyosin (Tm) with actin associated with the regulatory states of the muscle thin filament, we used luminescence resonance energy transfer (LRET) between Tb(3+) as a donor and rhodamine as an acceptor. A novel Tb(3+) chelator, S-(2-nitro-5-thiobenzoate)cysteaminyl-DTPA-Cs124, was synthesized, which specifically labels Cys groups in proteins. With the Tb chelate as the donor and tetramethylrhodamine-5-maleimide as the acceptor, both bound to specific Cys groups of Tm, we obtained 67 A as the distance between Tm's across the actin filament, a much shorter value than that obtained from structural studies (72-86 A). The difference appears to be due to submillisecond motion associated with Tm flexibility, which brings the probes closer during the millisecond lifetime of the donor. Ca(2+) did not change the energy transfer with the reconstituted thin filament, but myosin subfragment 1 decreased the transfer, consistent with either a 5-6 A increase in distance or, more likely, a decrease in flexibility.     

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.     

Distance measurements between metal-binding sites of calmodulin and from these sites to Cys-133 of troponin I in the binary complex

Distances between the four Ca2+-binding sites of calmodulin (CaM) have been measured by fluorescence energy transfer techniques using Eu3+ and Tb3+ as energy donors and a number of other lanthanide ions (Ln3+) as acceptors. It was shown previously that lanthanide ions preferentially bind to sites I and II of CaM with an affinity higher than that for sites III and IV (Kilhoffer, M.-C., Demaille, J. G., and Gerald, D. (1980) FEBS Lett. 116, 269-272; Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., and Gergely, J. (1982) Eur. J. Biochem. 124, 7-12). Thus upon direct excitation with a laser the luminescence lifetimes of Eu1Ln1CaM and Tb1Ln1CaM provide information on the distance between sites I and II. On the other hand, since Tb3+ ions bound to sites III and IV are sensitizable through tyrosine residues, lifetime measurements of Tb2Ln2CaM excited by UV light yield the distance between sites III and IV. Both pairs of sites were found to be separated by a distance of 1.05 +/- 0.07 nm. Binding of Ca2+ to sites III and IV does not alter the distance between sites I and II. We have also attached a chromophoric label, dimethylaminophenylazobenzene, to Cys-133 of skeletal troponin I and carried out distance measurements on its complex with CaM by both direct and indirect excitation. The averaged distances from sites I and II in the N-terminal half and from sites III and IV in the C-terminal half of the CaM molecule to the label on troponin I are 2.7 and 2.5 nm, respectively.     

Crystal structures and spectroscopic characterization of galactitol complexes of trivalent lanthanide and divalent alkaline earth chlorides

Crystal structures and FT-IR spectra of metal ion-galactitol (C6H14O6, the ligand here abbreviated as L) complexes: 2LaCl3*C6H14O6*10H2O and SrCl2*C6H14O6 complexes are reported. Crystal data of lanthanide chlorides (La3+, Nd3+, Sm3+, Eu3+, Tb3+)-galactitol complexes and alkaline earth chlorides (Ca2+, Sr2+)-galactitol complexes published earlier are summarized. Unlike other lanthanide ion-galactitol complexes (2MCl3*C6H14O6*14H2O), lanthanum ions give rise to two different structures: LaCl3*C6H14O6*6H2O (LaL1) and 2LaCl3*C6H14O6*10H2O (LaL2). Sr2+-galactitol complexes also crystallized with two structures: SrCl2*C6H14O6*4H2O (SrL1) and SrCl2*C6H14O6 (SrL2). These metal ions thus give different coordination structures with galactitol. The crystal structures and FT-IR spectra of lanthanide ion and alkaline earth ion-galactitol complexes were integrated to interpret the coordination modes of different metal ions. Similar IR spectra demonstrate the same coordination modes of the complexes.     

A gadolinium(III) complex of a carboxylic-phosphorus acid derivative of diethylenetriamine covalently bound to inulin, a potential macromolecular MRI contrast agent

A novel conjugate of a polysaccharide and a Gd(III) chelate with potential as contrast agent for magnetic resonance imaging (MRI) was synthesized. The structure of the chelate was derived from H5DTPA by replacing the central pendant arm by a phosphinic acid functional group, which was covalently bound to the polysaccharide inulin. On the average, each monosaccharide unit of the inulin was attached to approximately one (0.9) chelate moiety. The average molecular weight is 23110 and the average number of Gd3+ ions per molecule is 24. The ligand binds the Gd3+ ion in an octadentate fashion via three nitrogen atoms, four carboxylate oxygen atoms, and one P-O oxygen atom, and its first coordination sphere is completed by a water molecule. This compound shows promising properties for application as a contrast agent for MRI thanks to a favorable residence lifetime of this water molecule (170 ns at 298 K), a relatively long rotational correlation time (866 ps at 298 K), and the presence of two water molecules in the second coordination sphere of the Gd3+ ion. Furthermore, its stability toward transmetalation with Zn(II) is as high as that of the clinically used [Gd(DTPA)(H2O)]2-.     

Luminescence studies of Tb3+ bound to the high affinity sites of the Ca2+-ATPase of sarcoplasmic reticulum

Direct excitation of lanthanide luminescence with a pulsed dye laser has been used to probe the molecular environment of the high affinity sites of the sarcoplasmic reticulum Ca2+-ATPase. The direct excitation spectrum of Tb3+ bound to these sites has been determined and a luminescence lifetime of approximately 1 ms measured. Measurements of the difference in lifetime of the Tb X ATPase complex in H2O and D2O indicate that there are approximately 2 H2O molecules in the first coordination sphere of Tb3+ bound at the high affinity sites of the ATPase. The results are compared with the properties of Tb3+ binding to high affinity sites of other Ca2+ binding proteins. The binding constant of Tb3+ to the ATPase is in the range of 0.3-5.0 X 10(8) M-1 as inferred from the KI for inhibition of ATP hydrolysis, in agreement with a previous report (Highsmith, S. R., and Head, M. R. (1983) J. Biol. Chem. 258, 6858-6862). The values of the Ca2+ binding constant (approximately 2 X 10(6) M-1) and the cooperative nature (n = 1.9) of Ca2+ protection of Tb3+ inhibition indicate that Tb3+ and Ca2+ compete for the high affinity sites of the ATPase. The results demonstrate that directly-excited Tb3+ luminescence provides unique information on the environment of the Ca2+ binding-transport sites of the SR ATPase.     

Thiol-reactive luminescent chelates of terbium and europium

Thiol-reactive lanthanide complexes have been synthesized that are luminescent when bound to terbium and/or europium. The complexes consist of a diethylenetriaminepentaacetate (DTPA) chelate covalently joined through one amide bond to a chromophore, carbostyril 124, and via a second amide bond to a maleimide, bromoacetamide, or pyridyldithio moiety. Site-specific attachment and characterization of the complexes attached to DNA-activating protein NtrC, to various sites on myosin, or to DNA are presented. The compounds coordinate a surprisingly large number of ligation sites of terbium when a hydrazide spacer is used between the chelate and thiol-reactive moiety, although this extra ligation can cause quenching when europium is used. Synthesis is a simple two- or three-step reaction, and purification is straightforward. The compounds should be useful as nonisotopic replacements, as long-lifetime probes in imaging, and as donors in luminescence resonance energy transfer. They are examples of a wide class of chelates that can be made conjugatable via readily available hetero- or homo-bifunctional linkers.     

Interactions between metal ions and carbohydrates. Syntheses and spectroscopic studies of several lanthanide nitrate-D-galactitol complexes

Two complexes of neutral D-galactitol (C(6)H(14)O(6), G) with terbium nitrate, TbGN(I) and TbGN(II), and one complex with samarium nitrate SmGN were synthesized and characterized. From IR, FIR, THz and luminescence spectra the possible coordinations were suggested, and the single-crystal X-ray diffraction results confirm the spectroscopic conclusions. In TbGN(I) (Tb(NO(3))(3)·C(6)H(14)O(6)·3H(2)O), the Tb(3+) is 9-coordinated with three water molecules and six OH groups from two D-galactitol molecules. Nitrate ions do not coordinate to metal ions, which is different from other reported lanthanide nitrate-D-galactitol complexes. In TbGN(II) and SmGN (Ln(NO(3))(3)·C(6)H(14)O(6)), Ln(3+) is 10-coordinated with six OH groups from two D-galactitol molecules and four oxygen from two bidentate nitrate ions, and one nitrate ion is hydrogen bonded. No water exists in the structures. D-Galactitol molecules provide their 1-, 2- and 3-hydroxyl groups to coordinate with one metal ion and their 4-, 5- and 6-hydroxyl groups to coordinate with another metal ion in the three structures. There is still a new topological structure that can be observed for lanthanide-d-galactitol complexes, which indicates that the coordinations between hydroxyl groups and metal ions are complicated.     

A high-throughput screen for identifying transmembrane pore-forming peptides

We have developed a visual microwell plate assay for rapid, high-throughput screening for membrane-disrupting molecules such as de novo designed pore formers, antibiotic peptides, bacterial toxins, and lipases. The detectability is based on the strong fluorescence emission of the lanthanide metal terbium(III) (Tb(3+)) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb(3+) is not strongly fluorescent alone, the binary complex emits bright green fluorescence when irradiated with uv light. For the microwell plate assay, we prepared unilamellar phospholipid vesicles that had either Tb(3+) or DPA entrapped and the opposite molecule in the external solution. Disruption of the membranes allows the Tb(3+)/DPA complex to form, giving rise to a visibly fluorescent solution. In plates with 20-microl wells, the lower limit of visual detectability of the Tb(3+)/DPA complex in solution was about 2.5 microM. The lower limit of detectability using vesicles with entrapped Tb(3+) or DPA was about 50 microM phospholipid. We show that the membrane-disrupting effect of as little as 0.25 microM or 5 pmol of the pore-forming, antibiotic peptide alamethicin can be detected visually with this system. This sensitive, high-throughput assay is readily automatable and makes possible the visual screening of combinatorial peptide libraries for members that permeabilize lipid bilayer membranes.     

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

Effects of terbium chelate structure on dipicolinate ligation and the detection of Bacillus spores

Terbium-sensitized luminescence and its applicability towards the detection of Bacillus spores such as anthrax are of significant interest to research in biodefense and medical diagnostics. Accordingly, we have measured the effects of terbium chelation upon the parameters associated with dipicolinate ligation and spore detection. Namely, the dissociation constants, intrinsic brightness, luminescent lifetimes, and biological stabilities for several Tb(chelate)(dipicolinate)_x complexes were determined using linear, cyclic, and aromatic chelators of differing structure and coordination number. This included the chelator array of NTA, BisTris, EGTA, EDTA, BAPTA, DO2A, DTPA, DO3A, and DOTA (respectively, 2,2′,2″-nitrilotriacetic acid; 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N,N′,N′-tetraacetic acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid; diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid; 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Our study has revealed that the thermodynamic and temporal emission stabilities of the Tb(chelate)(dipicolinate)_x complexes are directly related to chelate rigidity and a ligand stoichiometry of x = 1, and that chelators possessing either aromaticity or low coordination numbers are destabilizing to the complexes when in extracts of an extremotolerant Bacillus spore. Together, our results demonstrate that both Tb(EDTA) and Tb(DO2A) are chemically and biochemically stable and thus applicable as respectively low and high-cost luminescent reporters for spore detection, and thereby of significance to institutions with developing biodefense programs.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Luminescence study of G-quadruplex formation in the presence of Tb3+ ion

The interactions of Tb3+ with the quadruplex-forming oligonucleotide bearing human telomeric repeat sequence d(G(3)T(2)AG(3)T(2)AG(3)T(2)AG(3)), (htel21), have been studied using luminescence spectroscopy and circular dichroism (CD). Enhanced luminescence of Tb3+, resulting from energy transfer from guanines, indicated encapsulation of Tb3+ ion in the central cavity of quadruplex core. The ability of lanthanide ions (Eu3+ and Tb3+) to mediate formation of quadruplex structure has been further evidenced by the fluorescence energy transfer measurements with the use of oligonucleotide probe labeled with fluorescein and rhodamine FRET partners, FAM-htel21-TAMRA. The CD spectra revealed that Tb3+/htel21 quadruplex possesses antiparallel strand orientation, similarly as sodium quadruplex. Tb3+ binding equilibria have been investigated in the absence and the presence of competing metal cations. At low Tb3+ concentration (8 microM) Tb3+/htel21 quadruplex stability is very high (5 x 10(6) M(-1)) and stoichiometry of 5-7 Tb3+ ions per one quadruplex molecule is observed. Luminescence and CD titration experiments suggested that the cavity of quadruplex accommodates two Tb3+ ions and the remaining Tb3+ ions bind probably to TTA loops of quadruplex. Higher concentration of Tb3+ (above 10 microM) results in the excessive binding of Tb3+ ions that finally destabilizes quadruplex, which undergoes transformation into differently organized assemblies. Such assemblies (probably possessing multiple positive charge) exhibit kinetic stability, which is manifested by a very slow kinetics of displacement of Tb3+ ion by competing cations (Li+, Na+, K+).