Synthesis of indole-containing diheteroarylethenes. New probes for photochromic FRET (pcFRET)

This paper reports a synthesis of novel diheteroarylethenes functionalized for coupling to biomolecules starting from indole derivatives. The strategy is based on the derivatization at the N-atom in the indole substructure. TBDMS protection proved to be superior over BOC protection schemes, leading to higher yields in the overall synthesis. The suitability of the new derivatives as acceptors for pcFRET was calculated for selected donors


Introduction
Photochromism is the result of reversible photoisomerization between two isomers that have distinct absorption spectra.The two isomers differ from one another not only in their absorption spectra but also in other physical and chemical properties. 1 Among the different photochromic compounds diheteroarylethenes are excellent candidates for optoelectronic devices due to their fatigue resistance and thermally irreversible conversion. 2 Diheteroarylethenes undergo isomerization from an open to a closed form (scheme 1) upon irradiation with UV light.Visible light converts the closed form back into the original open form.Symmetric indole-or pyrrole containing diheteroarylethenes give rise to thermally unstable closed form isomers.By replacing one indole with a furane, thiophene or benzothiophene group, the asymmetric diheteroarylethene becomes thermally stable in the closed form.A number of methyl indole derivatives have been introduced by Irie and coworkers 3 and the resulting photochromic compounds display interesting optical properties which makes them suitable as acceptors for energy transfer for a variety of fluorophore donors.

Scheme 1
FRET (Förster Resonance Energy Transfer) is a physical process by which energy is transferred non-radiatively from an excited molecular fluorophore (donor) to another chromophore (acceptor) via long-range dipole-dipole coupling.The FRET acceptor need not be fluorescent, but must fulfill the requirements of having an absorption spectrum overlapping the emission spectrum of the donor with the respective transition moments in a favorable, i.e. nonorthogonal, relative orientation.We have shown previously that diheteroarylethenes can be used as acceptors for photochromic Förster Resonance Energy Transfer (pcFRET), 4,5 a technique developed to perform the quantitative determination of FRET in vivo.The photochromic compound is converted from a colorless open form to a FRET-competent acceptor closed form upon irradiation with ultraviolet (UV) light.The open form lacks absorbance in the visible range.Thus, the overlap with the emission of the donor is negligible.The closed form has an absorption band overlapping the emission band of the donor, which can be switched back to the open, nonoverlapping form by exposure to visible light.Multiple "on"/"off" FRET cycles can be generated by alternating exposure to UV and visible light.
Application of photochromic compounds as pcFRET acceptors in biology requires that they contain functional groups that can be used for their conjugation to (bio)molecules.Indole derivatives offer the opportunity for preparing a functionalized substituent on the N atom.Here we report the synthesis of asymmetric diheteroarylethenes bearing a removable protecting group at this position.
The diheteroarylhexafluorocyclopentenes are usually obtained by a reaction involving nucleophilic attack of a heteroaryllithium on octafluorocyclopentene, followed by elimination of fluoride groups. 2,6 ith indole as one of the heteroaryl functionalities, the heteroaryllitium species can be formed by halogen-lithium exchange reaction of the N-protected 3-haloindoles.Synthesis of these haloindoles is usually performed by electrophilic substitution.N-Protection of the indoles can be carried out before or after the halogenation step.As a result, the requirements of the protecting group are dictated by the lithium-halogen exchange reaction conditions.The synthesis and application of these N-protected indoles is well documented: 7,8 alkyl, silyl, alkoxymethyl, acyl and other protecting groups have been used.Here we investigated their use for reaction with perfluorocyclopentene.
The first class of protective group investigated in this synthetic approach was the tbutoxycarbonyl group (BOC) (Scheme 2).3-Iodo-2-methylindole 2 was obtained by iodination of 2-methylindole 1 9, 10 which was followed by protection of the N-position of the indole nucleus to give 3.In order to assess the best reaction conditions, different stoichiometric relationships between n-butyllithium and the alkylating reagent CH 3 I were tested (reaction not shown in Scheme).The best yields (65-86%) were obtained when 2 equivalents of n-butyllithium and 1.5-3 equiv of CH 3 I were used at -30 ºC.Lithiation of the 3-iodoindole 3, followed by reaction with excess octafluorocyclopentene led to compound 4 in 42% yield.However, when a similar substitution reaction of the lithiated indole with compound 7 was attempted, the reaction proceeded sluggishly and only 29% of the desired product (5) was isolated, recovering 50% of starting material.When excess n-butyllithium was used in the halogen-metal exchange reaction, the desired product was isolated in 20% yield.In addition, the side product from reaction of unreacted n-butyllithium with 7 gave 6 in 48% (based on 7).

Scheme 2
TBDMS was applied in an alternative protection strategy (Scheme 3).Lithiation of compounds 11 and 12 with n-butyllithium at -78 ºC, followed by reaction with 7 or 16 (11 only) rendered the desired diheteroarylethenes 13 -15 in reasonable yields (Scheme 4).These compounds already contain the photochromic moiety and bear orthogonally protected phenolic (OH; 14 only) and indolic (NH) functionalities suitable for further derivatizations.

Scheme 4
Deprotection of the indole-nitrogen proceeded smoothly with TBAF (Scheme 5).Immediate reaction with NaH followed by alkylation with ethyl bromoacetate in DMF rendered 18, which by hydrolysis gave the final product 19. 12,13 ompound 19 contains a carboxylic acid group that can be further activated for conjugation with amine groups.Upon irradiation with UV, the color of the solutions went from colorless to red (compounds 5, 15, and 16) or blue (compound 14).The effect of substituent groups on the N atom was evaluated by comparison of the optical properties of the BOC, TBDMS, CH 3 , 2 and CH 2 COOR derivatives.The closed form (Figure 1 b) displayed an absorption maximum at ca. 560 nm for the TBDMS, CH 3 and CH 2 COOR substituents.The maximum was shifted to 511 nm for the less electron rich BOC group.
Introduction of a methoxy substituent is known to affect the absorption maxima and extinction coefficient values, depending on its position on the benzothiophene ring of a diarylethene. 14A methoxy group on position 6 (compound 15) exerted a negligible change in the absorption maximum and displayed a slight increase in ε compared to 13.On the other hand, the introduction of a methoxy group in the position 5 of the indole moiety (compound 14) introduced a red-shift of ca.40 nm compared to compound 13 and a decrease of ε to 9200 M -1 cm -1 .
Indole derivatives as acceptors for pcFRET.The modulation of the emission of a fluorescent donor by pcFRET is based on the difference in the absorption properties of the acceptor in its different photochromic forms.Compounds 5, 13-15 were evaluated as switchable acceptors.

Conclusions
The synthetic scheme using TBDMS as protective group was superior over the use of BOC for the preparation of functionalized, indole derived asymmetric diheteroarylethenes.The substitution on the indole ring at the N-atom had minor effects on the optical properties except for the BOC derivatives.Methoxy groups at position 5 on the indole moiety induced a red-shift of the absorption of the closed form to 600 nm and a decrease in ε.All photochromic compounds prepared in this work constitute good acceptors that can switch on and off the FRET process in pcFRET.electronic timer for exposure time control (Lumatec GmbH, Munich, Germany).Monochromic light was obtained by passing the light a band-pass filter (∆λ 1/2 =10 nm).The photoconversions were determined with an Ocean Optics fiber optics spectrometer system with a cuvette holder specially modified to allow simultaneous photoconversion and spectral monitoring.Excitation for absorption and fluorescence was with a DT1000A deuterium/tungsten lamp, and detection was with a SD2000 series dual fiber optics spectrometer optimized for detection in the 250-800 nm spectral region.

Experimental Section
FRET Methods., the critical Förster distance for 50% FRET efficiency, is defined by units, nm 6 ) where Φ is the quantum yield of the donor in the absence of acceptor, n is the refractive index of the medium, κ 2 is the orientation factor between donor and acceptor (here assumed to be 2/3, the value corresponding to rapid and isotropic reorientation of donor and acceptor during the excited state), and J is the spectral overlap integral between donor and acceptor, given by , where F λ is the normalized donor fluorescence spectrum and ε λ is the wavelength-dependent molar extinction coefficient (M -1 cm - 1 ) of the acceptor.For an isolated donor-acceptor pair, the FRET efficiency E varies according to Materials.2-methyl-1H-indole (1), 5-methoxy-2-methyl-1H-indole (8), are commercially available and were obtained from Aldrich Chem.Co. Compounds 7 15 and 16 14 were prepared as previously described.

Figure 2
displays the absorption spectra corresponding to the open and closed forms of compound 15 and the emission spectrum of Lucifer yellow (LYC).Compound 15 displays an effective spectral overlap between its absorption and the emission of the donor (LYC) only in the closed form.

Figure 2 .
Figure 2. Spectral overlap of the open and closed forms of 15 and LYC.Open (full circles) and closed (empty circles) form extinction coefficients of 15 correspond to the right axis scale and the emission intensity of LYC (full squares) is given on the left axis.

Table 1 .
Optical properties of compounds 5

, 13-15
434Photochromic properties.All synthesized diarylethenes underwent reversible photochromic reactions in cyclohexane upon alternating exposure to UV (340 nm) and visible (546 nm) light.Compounds 5, 13 and 14 displayed two isosbestic points.Compound 5 at 299 and 316 nm; compound 13 at 313 and 321 nm, and compound 14 at 311 and 332 nm.On the other hand, compound 15 had one isosbestic point at 281 nm and compound 18 lacked an isosbestic point.