Synthesis and structure optimization of double (fluorescent and spin) sensor molecules

Synthesis and fluorescence properties of stable nitroxide free radicals ( 10a, 11a, 12a, 14a, 20a, 21a ) and their amine ( 10b, 11b, 12b, 14b, 20b, 21b ) precursors covalently linked to dansyl or 3-and 4-aminophthalimide are reported. The best intramolecular quenching is achieved when the


Introduction
Fluorescence and spin double sensors are important probe molecules for detecting free radicals both in condensed- [1][2][3][4] and gas-phases 5 .Their sensing ability is based on the energy transfer from a donor moiety (fluorophore) to an acceptor moiety (nitroxide) which results in quenched fluorescence.][8] Absence of acceptor in the diamagnetic derivatives of these probe molecules, exhibit strong fluorescence.Utilizing these probes, Reactive Oxygen Species (ROS) production can be followed on the basis of either fluorescence quenching or the EPR detectable appearance of nitroxide.In case of biological application other requirements arise: to avoid the overlapping with background emission of intrinsic fluorophores, water solubility and permeability through membranes.Applications also require a large difference between the fluorescence emission of the amine and the corresponding nitroxide, in order to assure sensitive detection of ROS.Recently we have developed a sensor molecule for biological application, called DanePy 1 1b, which is readily oxidized by ROS to nitroxide 1a (Scheme 1).

Scheme 1
Similar oxidation reaction was also observed earlier in vivo. 9The diethylaminoethyl side-chain in DanePy 1b ensures water solubility and penetration into chloroplasts. 10In this paper we discuss the role of spacer group on quenching of fluorescence as well as extension of this idea to other aminophthalimide fluorophores to obtain more sensitive double sensors.

Results and Discussion
In our previous studies we experienced the advantage of a protonable amino group in spacer to increase of solubility in aqueous media.We achieved this by inserting of piperazine, or 1-(2aminoethyl)piperazine as spacer group between nitroxide and fluorophore in double sensors.This could be accomplished by alkylating piperazine with allylic bromide 11 2 in CHCl 3 in the presence of K 2 CO 3 to give mixture of the monoalkylated compound 3 and the dialkylated compound 4. The N- [2-(1-piperazidinyl)ethyl]phthalimide 5 12 could be alkylated on the secondary nitrogen atom under the above conditions to yield compound 6.Treatment of phthalimide derivative 6 with methylamine 13 in ethanol allowed the mild deprotection of the terminal amino group to give compound 7 (Scheme 2).
However, comparing the ratios of fluorescence emission maxima of amines 10-12b versus nitroxides 10-12a in case of compound 10, 11 became less advantageous than 12b/12a ratio (Table 1).The introduction of a long spacer between the donor and the acceptor moieties decreased the rate of quenching of fluorescence, because the rate of Coulombic energy transfer inversely proportional to the sixth power of distance between donor and acceptor.While electron transfer or electron exchange rate decrease exponentially with increasing donor-acceptor distance. 16To get better double sensor molecules, i. e. to achieve better ratio of emission maxima of amine versus nitroxide we investigated compound 13. 17 The sodium salt of 13 was alkylated in dimethylformamide/THF mixture with freshly released 2-(diethylamino)ethyl chloride to give compound 14a, which was then reduced to 14b with Fe powder in AcOH (Scheme 4).
Fluorescence emission maxima ratio of compounds 14b and 14a were the highest among the investigated dansyl derivatives (Table 1).
This idea can be extended to other donor-acceptor pairs as we demonstrate in the case of 3-and 4-aminophthalimide as donor and 1-oxyl-2,2,6,6-tetramethyl piperidine as acceptor.We chose 3and 4-aminophthalimides because beyond their fluorescence properties they exhibited the ability of recognizing CG Watson-Crick base pair, as reported very recently. 18
In conclusion, the best double sensor reagents among the ones we tested were those with a minimal acceptor-donor distance.However, side-chains may prove to be advantageous in further biological applications.

Experimental Section
General Procedures.Melting points were determined with Boetius micro melting point apparatus and are uncorrected.Elemental analyses (C, H, N, S) were performed on Fisons EA 1110 CHNS elemental analyser.Mass spectra were recorded on a VG TRIO-2 instrument in EI mode (70 eV, direct inlet) or with thermospray technique (TSP).Samples were analyzed in bypass mode.10 µL of the sample solution in MeOH was introduced via the thermospray interface.The mobile phase was MeOH/H 2 O (1:1) containing 0.1 M NH 4 OAc.The capillary tip temperature was 230 °C, the electrode voltage was 180 V and source temperature 210 °C.The ESR spectra were obtained from 10 -5 molar solution (CHCl 3 ), using Bruker ECS-106 spectrometer.All monoradicals exhibit three equidistant lines with a N = 14.3-14.8G. Fluorescence emission spectra were recorded with Quanta Master QM-1 (Photon Technology International Inc.) using 345 nm excitation wavelength and 1 nm excitation and emission slits.The fluorescence emission peak intensities were normalised by concentration.Flash column chromatography was performed on Merck Kieselgel 60 (0.040-0.063 mm).TLC was carried out on commercially prepared plates (20x20x0.02cm) coated with Merck Kieselgel GF 254 .Compounds 9, 15, 16, 17 were purchased from Aldrich.Compounds 1, 1 5, 12 11 17 were prepared according to published procedures.

Synthesis of diamagnetic dansyl and phthalimidyl derivatives. General procedure
To a solution of nitroxide 10a or 11a or 12a or 14a or 18 or 19 (2.0 mmol) in glacial acetic acid (10 mL) Fe powder (560 mg, 10.0 mmol) or (1.12 g, 20.0 mmol in case of 18 or 19) was added and the mixture was warmed up to 50 °C until the reaction started and the reaction was stirred for 30 min at r.t.After diluting water (30 mL), the solution was decanted from iron residue, and the solution was made alkaline (pH = 9) by adding solid K 2 CO 3 .The reaction mixture was filtered off, the filtrate was extracted with CHCl 3 (3 x 40 mL), and the separated organic phases were combined, dried (MgSO 4 ), filtered and evaporated in a vacuum.Flash column chromatography with CHCl 3 /MeOH as eluent afforded the title amines 10b or 11b or 12b or 14b or 20b or 21b as yellow-green solids or oils.