No abstract

Complementary synthetic routes to a new class of near IR fluorophores are described. These allow facile access (four synthetic steps) to the core fluorophore and substituted derivatives with emissions between 740 and 780 nm in good quantum yields.Surprisingly few fluorescent probes emit in the near-IR region with high quantum yields. 1, 2 Such compounds are valuable for intracellular imaging because autofluorescence in cells tends to obscure emissions at wavelengths below approximately 550 nm, but this factor becomes less of an issue at longer wavelengths. Probes that emit in the 750 -900 nm region are, therefore, relatively easy to visualize in vivo. 3 Cyanine dyes are currently the most widely used probes for this wavelength region, but their quantum yields and photostabilities are sub-optimal. 4 Thus there is need for new fluorescent probes that emit efficiently above 750 nm. Dyes based on the 5-6-5 fused BODIPY ring system A are popular in biotechnology because they tend to have relatively sharp fluorescence emission characteristics, and high quantum yields. 5 However, the emission wavelengths of most BODIPY dyes are between 530 and 630 nm, and this is sub-optimal for intracellular or tissue imaging. Previous work by Burgess et al ,6-8 and others, 9,10 successfully explored a modification strategy for shifting BODIPYfluorescence emissions to the red ( Figure 1). Adaptation of the core fluorophore scaffold by intramolecular B-O ring formation to produce the 6,6-5-6-5-6,6 ring system B gave a ca 65 nm bathochromatic shift (compare C and D), 11 but the fluorescence emission wavelengths were less than 700 nm. 12O'Shea and co-workers have demonstrated that BF 2 chelated tetraaryl-substituted azadipyrromethenes E, fluoresce with wavelength maxima between 670-720 nm with paraoriented electron donating groups being particularly effective in enhancing the red-shifted fluorescence, eg F (Figure 2). 13-19 This is close to the 750 -1,400 nm region at which light permeation through tissue is most effective. Subsequently, it was shown that derivatives with extended aromatic groups do emit in near-IR region. 20 The premise of this paper is that it would be advantagous to "hybridize" the structure B and E to shift their emissions beyond 750 nm. On the basis of the structures shown in Figure 1 it was therefore logical to introduce an intramolecular B,O-chelate on a tetraaryl aza-BODIPY scaffold eg G. The studies described here feature compound 1 of this type; it emerges that they can emit in this region.Scheme 1 shows one approach to the synthesis of the target class 1 from the precursors 2a-d which can be synthesized in four steps from commercially available materials. 21 A solid state X-ray structure determination of 2a illustrated that both 2-MeOC 6 H 4 rings lie out of the plane of the central chromophore with torsion angles of 42.7° and 61.4° (Supporting Information). Compounds 1 were obtained from the corresponding 2 by treatment with boron tribromide in dichloromethane. We anticipated that demethylation of the...

A molecule that transfers energy through bonds from a donor to an acceptor was prepared with a pH sensitive donor function (fluorescein). At pH values above 6.5, minimal energy transfer occurred, and the probe emits green fluorescence (ca 520 nm) when excited at the donor (488 nm). Below pH 6 however, energy transfer is efficient hence excitation at the donor causes emission at the acceptor part (600 nm). This probe was used to image a conjugate of the probe with bovine serum albumin that was imported into endosomes or in the cytosol using the non-covalently bound carrier, Pep-1, at 37 and 4 °C, respectively. The more acidic environment of the endosomes was conspicuous from the red fluorescence of the probe.

This paper concerns the development of water-compatible fluorescent imaging-probes with tunable photonic properties that can be excited at a single wavelength. Bichromophoric cassettes 1a -1c consisting of a BODIPY donor and a cyanine acceptor were prepared using a simple synthetic route, and their photophysical properties were investigated. Upon excitation of the BODIPY moiety at 488 nm the excitation energy is transferred through an acetylene bridge to the cyanine dye acceptor, which emits light at approximately 600, 700, and 800 nm, ie with remarkable dispersions. This effect is facilitated by efficient energy transfer that gives a 'quasiStokes' shift of between 86 -290 nm opening a huge spectral window for imaging. The emissive properties of the cassettes depend on the energy transfer (ET) mechanism: the faster the transfer, the more efficient it is. Measurements of rates of energy transfer indicate that a through-bond energy transfer takes place in the cassettes 1a and 1b that is two orders of magnitude faster than the classical through-space, Förster, energy transfer (in the case of cassette 1c, however, both mechanisms are possible, and the rate measurements do not allow us to discern between them). Thus the cassettes 1a -1c are well suited for multiplexing experiments in biotechnological methods that involve a single laser-excitation source. However, for widespread application of these probes their solubility in aqueous media must be improved. Consequently, the probes were encapsulated in calcium phosphate/silicate nanoparticles (diameter ca 22 nm) that are freely dispersible in water. This encapsulation process resulted in only minor changes in the photophysical properties of the cassettes. The system based on cassette 1a was chosen to probe how effectively these nanoparticles could be used to deliver the dyes into cells. Encapsulated cassette 1a permeated Clone 9 rat liver cells where it localized in the mitochondria and fluoresced through the acceptor part, ie red. Overall, this paper reports readily accessible, cyanine-based through-bond energy transfer cassettes that are lypophilic but can be encapsulated to form nanoparticles that disperse freely in water. These particles can be used to enter cells and to label organelles.

Fluorescent DNA-labeling cassettes are designed to have a common absorbing chromophore matched to a single exciting laser wavelength, but up to four different emitters. Experiments reported here have examined the energy-transfer rates and fluorescence polarization characteristics for two different types of cassette, involving three distinct relative orientations of the donor and acceptor transition moments and the axis of the rigid linker. Energy-transfer times range from <200 fs to approximately 20 ps, the fastest transfer times occurring when the transition moments of the donor and acceptor species are aligned parallel to the linker axis. Experimental evidence is presented that supports a through-bond energy-transfer mechanism, in contrast with a commercial DNA-labeling agent, which exhibits much slower transfer times controlled by FRET. These rigid cassettes also exhibit polarized fluorescence from the acceptor species, so that this particular type of DNA-labeling probe has some of the advantages of single-molecule probes such as rhodamine and coumarin dyes.