Long-lived Probes

Rhenium (I) complexes of the general structure depicted in Fig. 6.7 have been shown to display remarkable photo luminescent properties since the 1970s [44, 45]. 

These emission features are associated with the lowest lying MLCT excited state involving a metal-centered dn-type orbital and a k orbital centered on the a-diimine ligand. These transitions appear in the visible range of the spectrum. Noticeably, characteristics of excited states such as emission quantum efficiency, lifetime and, redox potential are greatly affected by the ancillary ligands, solvent polarity and other environmental factors [46]. 

These photophysical properties have been exploited to design chemical sensors for O2 [47], cations [46] and anions [48]. Several rhenium(I) polypyridine biotin compounds were shown to display up to a 3-fold enhancement of their emission intensity upon binding to the protein avidin [49]. 

Interest was recently directed to the conjugation of this class of complexes with proteins, in order to benefit from their exceptional photophysical properties. 

Lakowicz et al. described the first protein conjugable luminescent rhenium(I) complex 27 that was covalently linked to the proteins BSA and bovine IgG by reachon of some of their lysine residues after activation of its carboxylic add function by a mixture of DCC and NHS (see Chapter 8, Section 8.4.7) [50]. 

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An explanation of the discrepancies was offered by the results from some experiments made by Johannsson et al. [30], employing a more long lived probe, Crfbpy) (t 25 /is), in the AOT-alkane-water system. From the observation of two decay processes, well separated in time as shown in Fig. 7, the authors concluded that small clusters of reverse micelles were present in the microemulsions. The initial fast process, the intramicellar quenching, occurs on the submicrosecond time scale and appears only as an initial drop since it is not resolved on the time scale used with Cr(bpy)3. It is this part of the deactivation that is possible to monitor in normal TRLQ measurements with short-lived probes. The initial drop is followed by a second decay with a characteristic time of a few microseconds before the final, very slow deactivation occurs. The results suggest that the fast exchange... 

In this ch ter we have attempted to show die potential of time-resolved measurements for determination of the solution conformations of macromolecules. The determination of Conformational distributions appears to be a rather unique property of the time-resolved measurements. Most physical methods reveal only an average conformation. Additionally, the data contain information on the timescale of conformational changes. With the introduction of long-lived probes, conformational dynamics will be measurable on timescales from nanoseconds to tens of microseconds. 

Van den Bogaard P, Schimick C (1995) " °Ar/ Ar laser probe age of Bishop Tuff quartz phenocrysts substantiate long-lived silicic magma chamber at Long Valley, United States. Geology 23 759-762 Van Orman J.A., Grove T.L., Shimizu N (1998) Uranium and thorium diffusion in diopside. Earth Planet Sci Lett 160 505-519... 

Pyrene fluorophores are also used as probes. Derivatives of pyrene show /.max/ Xem 340/376 nm, e 4.3 x 104 M 1 cm-1, and environmental sensitivity, this fluorophore can be used to report on RNA folding [102]. Pyrene also displays a long-lived excited state (x > 100 ns), which allows for an excited pyrene molecule to associate with a pyrene in the ground state. The resulting eximer exhibits a red-shift in fluorescence intensity (A,em 490 nm). This characteristic can be used to study important biomolecular processes, such as protein conformation [103]. 

As discussed in Sect. 1.2.3, it is usually not possible to distinguish ESA from 2PA with Z-scan experiments if they are performed with only one excitation pulsewidth. However, since ESA is not an instantaneous process as is 2PA, the pump-probe technique can be successfully used to verify the origin of the nonlinearity for the spectral regions close to the main absorption band. Figure 7 illustrates how the influence of the ESA can be distinguished from the 2PA with pump-probe experiments. The curve labeled (1) shows an instantaneous 2PA response without ESA and the long-lived components of the transmittance change seen in (2) and (3) are due to ESA. 

Until very recently, studies of the use of luminescent lanthanide complexes as biological probes concentrated on the use of terbium and europium complexes. These have emission lines in the visible region of the spectrum, and have long-lived (millisecond timescale) metal-centered emission. The first examples to be studied in detail were complexes of the Lehn cryptand (complexes (20) and (26) in Figure 7),48,50,88 whose luminescence properties have also been applied to bioassay (vide infra). In this case, the europium and terbium ions both have two water molecules... 

Hemmila, 1988) (see Chapter 9, Section 9). The most commonly used lanthanides for this purpose are europium (Eu3+), terbium (Tb3+), and samarium (Sm3+). Proteins modified with DTTA and complexed with lanthanide metal ions form the basis for unique fluorescent probes possessing long lived signals upon excitation. 

Spectroscopically invisible carbenes can be monitored by the ylide method .92 Here, the carbene reacts with a nucleophile Y to form a strongly absorbing and long-lived ylide, competitively with all other routes of decay. Although pyridine (Py) stands out as the most popular probe, nitriles and thiones have also been used. In the presence of an additional quencher, the observed pseudo-first-order rate constant for ylide formation is given by Eq. 2.92,93 A plot of obs vs. [Q] at constant [Y ] will provide kq. With Q = HX, complications can arise from protonation of Y and/or the derived ylides. The available data indicate that alcohols are compatible with the pyridine-ylide probe technique. 

E Sacksteder, M. Lee, J. N. Demas, and B. A. DeGraff, Long lived, highly luminescent rhenium(I) complexes as molecular probes Intra- and intermolecular excited state interactions, J Am. Chem. Soc. 115, 8230-8238 (1993). 

Reticulum ATPase [105,106], Owing to the long-lived nature of the triplet state, Eosin derivatives are suitable to study protein dynamics in the microsecond-millisecond range. Rotational correlation times are obtained by monitoring the time-dependent anisotropy of the probe s phosphorescence [107-112] and/or the recovery of the ground state absorption [113— 118] or fluorescence [119-122], The decay of the anisotropy allows determination of the mobility of the protein chain that cover the binding site and the rotational diffusion of the protein, the latter being a function of the size and shape of the protein, the viscosity of the medium, and the temperature. 

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