Two-photon FRET pairs based on coumarin and DBD dyes †

The synthesis and photophysical properties of two new FRET pairs based on coumarin as a donor and DBD dye as an acceptor are described. The introduction of a bromo atom dramatically increases the two-photon excitation (2PE) cross section providing a 2PE-FRET system, which is also suitable for 2PE-FLIM.

Amongst various analytical techniques, the evaluation of uorescence signals is one of the most sensitive methods. 1 Therefore, uorescence spectroscopy has found a wide range of applications in biology, biochemistry, and clinical diagnostics.Besides classic steady state measurements that evaluate the uorescence intensity, methods utilizing the uorescence lifetime have gained growing interest in recent years.Both parameters are successfully applied in uorescence imaging.The pivotal element of these methods is the uorescent dye and its properties.Small uorescent organic molecules play, beside quantum dots (QD) 2 and luminescent lanthanide complexes, 3 the central role in current uorescence spectroscopy.The potential of a uorescent dye is determined by the combination of its photophysical parameters, such as uorescence quantum yield (F F ), molar extinction coefficient (3), uorescence lifetime (s F ), absorption and emission wavelength (l ABS , l EM ), and stability against photobleaching.Because the penetration depth of light in biological tissue is increased with increasing wavelength, high l ABS and l EM are desirable.However, a longwavelength absorption maximum is usually connected with low thermal and photochemical stability of the dyes.This problem can be circumvented by two-photon excitation (2PE), 4 which is based on the simultaneous absorption of two photons with lower energy instead of one photon with high energy.Consequently, the excitation wavelength l EXC is almost doubled in 2PE.The probability of the 2PE is described by its 2PE cross section s 2 .Unfortunately, s 2 is low for most organic molecules. 5n the other hand the applicability of many uorescence dyes suitable for 2PE is limited when considering the other photophysical properties.The combination of the outstanding properties of two uorescent dyes within one molecule can be achieved by a pair of dyes capable for Förster Resonance Energy Transfer (FRET). 6This phenomenon, in which one dye (the donor D) is excited and the energy is transferred to the other dye (the acceptor A), is observed if the emission spectrum of D and the absorption spectrum of A sufficiently overlap.Furthermore, the FRET efficiency is strongly distance-dependent (the distance with 50% FRET efficiency is called Förster distance R 0 ) and is inuenced by the relative arrangement of the transition dipole moments of D and A. A special type of FRET exists if the donor D is capable of 2PE.In this case, the uorescence of the donor D as well as the acceptor A can be observed at shorter wavelength than the excitation ("Anti-Stokes shi").Although some examples of 2PE-FRET pairs were reported in the literature, 7 the number of 2PE-FRET systems is rather limited.
In the last few years, we have developed a new class of uorescent dyes, whose structure is based on [1,3] [8][9][10][11][12][13][14] These dyes are characterised by large Stokes shis (Dl ¼ l EM À l ABS > 100 nm), combined with long uorescent lifetimes (s F > 20 ns) and exceptional bleaching stability.The 2PE cross-sections s 2 of DBD dyes are, however, rather low (vide infra).Recently, we reported on a rst FRET pair with DBD dyes as acceptor and 2,5-diphenyloxazol (PPO) as donor. 13Herein we wish to report on the synthesis and properties of new FRET pairs with coumarin derivatives as donor and DBD dyes as acceptor for 2PE-FRET application.
The coumarin chromophore was chosen because it is already known to be suitable for 2PE. 15The synthesis starts with the commercially available 6,7-dihydroxy-coumarin 1.The reaction with benzyl prop-2-ynoate 16 in the presence of catalytical amounts of DMAP afforded coumarin 2 with very good yield.
This type of cyclisation has already been described for other catechols 8,10,17 but never applied to coumarins.Because the introduction of halogen atoms regularly increases the 2PE crosssection, 2 was brominated with NBS to give 3-bromo-coumarin in quantitative yield.Subsequently, esters 2 and 4 were deprotected by catalytic hydrogenation to the carboxylic acids 3a,b (Scheme 1).The connection between coumarin and DBD chromophore was accomplished by a 1,2-diaminoethane linker.For this purpose, esters 3a,b were converted into amides 6a,b by reaction with commercially available N-Boc-1,2-diaminoethane 5, which were subsequently deprotected to give primary amines 7 (Scheme 2).
Finally, the FRET pairs 10a,b were prepared from amines 7a,b and acyl chloride 9, which is easily accessible from the known DBD acid 8 (Scheme 3).Next, we investigated the spectroscopic properties of compounds 10a,b in comparison with the starting compounds 2, 4, 8.The results in DMSO are summarized in Table 1 (for other solvents see ESI †).Of particular note are the large Stokes shi (133 nm) and the long uorescence lifetime (24.8 ns) of DBD dye 8 compared with coumarins 2 and 4. The successful FRET in compound 10a,b was shown by 3D-uorescence experiments, which are exemplarily outlined for 10a in Fig. 1.
The highly efficient FRET in compound 10a is stressed in Fig. 1C by (i) the strong quenching of the donor emission (compare to Fig. 1A) and (ii) the strong emission of the DBD acceptor under indirect excitation via the coumarin donor (compare to Fig. 1B).This result was to be expected, because the maximum possible distance between the chromophores (1.5-1.6 nm) is markedly below the Förster radius R 0 for this FRET pair (2.56 nm, for details see the ESI †).
Whereas the 2PE cross section s 2 is very low for DBD compound 8 and coumarin 2, the introduction of bromine in coumarin 4 signicantly enhances the s 2 value to 0.74 GM (see Table 1).
In order to explore potential applications of the 2PE-FRET pairs-sensor 10b in living cells, its uptake into such was studied in 2PE-uorescence lifetime imaging (2PE-FLIM) experiments.Insect salivary gland lobes were incubated for 15 min with 2 mM 10b and then, the tubular-like salivary ducts were imaged.Salivary ducts without 10b-loading displayed a comparatively low autouorescence when excited at 780 nm as expected for 2PE (Fig. 2A and E). 18Mainly the luminal cuticule and tracheae contributed to the autouorescence (Fig. 2B) and the corresponding uorescence lifetime distribution uctuated around 3 ns (Fig. 2F).In contrast, the uorescence intensity of 10b-loaded salivary ducts was up to one order of magnitude higher than that of unloaded ducts (Fig. 2C and E).Such a loading efficiency has previously been reported for other ionsensitive uorescent dyes. 19Here, the duct cells were stained strongly and 10b did not accumulate in specic cellular compartments.However, almost no uorescence could be observed in the nuclei and the duct lumen (Fig. 2D).This result is a prerequisite for successful intracellular recordings using the novel 2PE-FRET pairs presented.The sufficient 10b-loading into living cells could also be observed in the uorescence lifetime distribution, which was now shied to longer lifetimes around 5.5 ns (Fig. 2F).

Conclusions
Both single chromophores exhibit specic pros and cons.The 3bromocoumarin shows a sufficiently large 2PE cross section (0.74 GM) but has a low uorescence lifetime (1.3 ns) and a relatively short emission wavelength (436 nm).
By contrast, the DBD chromophore shows long emission wavelength (570 nm), large uorescence lifetime (23-25 ns) but very low 2PE cross section.The FRET pair 10b perfectly combines the advantages of these dyes.Aer long-wavelength two-photon excitation at 780 nm an efficient FRET takes place resulting in a long-lived emission at 570 nm.The applicability of 10b for 2PE-FLIM was demonstrated with the aid of living cells of insect salivary gland lobes.In this application the large contrast range of 1-7 ns is noteworthy.Currently, we are investigating synthetic routes to derivatives of 10b, which are suitable for coupling with various biomolecules.