Folding‐induced Fluorescence Enhancement in a Series of Merocyanine Hetero‐Folda‐Trimers

Abstract Many dyes suffer from fast non‐radiative decay pathways, thereby showing only short‐lived excited states and weak photoluminescence. Here we show a pronounced fluorescence enhancement for a weakly fluorescent merocyanine (MC) dye by being co‐facially stacked to other dyes in hetero‐folda‐trimer architectures. By means of fluorescence spectroscopy (lifetime, quantum yield) the fluorescence enhancement was explained by the rigidification of the emitting chromophore in the defined foldamer architecture and the presence of a non‐forbidden lowest exciton state in H‐coupled hetero‐aggregates. This folding‐induced fluorescence enhancement (FIFE) for specific sequences of π‐stacked dyes points at a viable strategy toward improved fluorophores that relates to the approach used by nature in the green fluorescent protein (GFP).

Fluorescence spectroscopy and quantum yield determination: Steady-state absorption spectra were recorded using a V770 UV-Vis spectrometer (JASCO Inc., Japan). Emission and excitation spectra were measured with a FLS980-D2D2-ST (Edinburgh Instruments Ltd., UK) fluorescence spectrometer and corrected against the photomultiplier sensitivity and the lamp intensity. All spectra were recorded at 293 K, if not stated otherwise, and the temperature was controlled by a sample holder with Peltier element. The fluorescence quantum yields (Φ fl ) were determined as average value of four different excitation wavelengths relative using N,N'-bis(2,6diisopropylphenyl)-1,6,7,12-tetraphenoxy-perylene-3,4:9,10-bis(dicarboximide) (Φ fl = 0.96 in chloroform) [S9] and Rhodamine 800 (Φ fl = 0.25 in ethanol) [S10] as standards under highly diluted conditions (OD ≤ 0.05) and magic angle conditions (54.7°). The fluorescence lifetimes were determined by Time Correlated Single Photon Counting (TCSPC) using a EPL510 pulsed diode laser ( exc = 505.8 nm) with a pulse width of 141.7 ps or a EPL485 pulsed laser diode ( exc = 479.7 nm) with a pulse width of 113.8 ps with an FLS980-D2D2-ST spectrometer (Edinburgh Instruments Ltd., UK) under magic angle conditions (54.7°). The fitting of the data was carried out using the Recursive-Fit-routine of the FAST software supplied by Edinburgh Instruments Ltd., Inc. which corrects against the instrument response function (IRF). The existence of an ensemble of inhomogeneously folded states with presumably different emission properties means the determined  rel can be described as the sum of the different quantum yields times the ratio of species excited : rel = ∑ i * i = open * open + folded * folded + ∑ n * n ⏟ other species (1) As we only observed emission which we attribute to a partially folded (open) or a completely folded form we assume any other possible species to be negligible. Therefore, the term can be simplified to: To minimize contamination of the spectrum with the other emissive species, we measured an emission map over a wide range of excitation wavelengths. On the next slide our approach is explained in detail for the partially folded form of RRB in CHCl 3 , but the same approach was used for the completely folded species of RRB in CHCl 3 /MCH 25:75 and for the partially folded species of the other trimers. Figure S1. Emission spectra of RRB at different excitation wavelengths using a) a visible PMT and b) a NIR PMT in CHCl3 at 293 K (c ≈ 0.1 μM).

S3
From these spectra we can see that the emission spectra at excitation wavelengths over 520 nm are almost identical, indicating that the amount of excited completely folded species is negligible at these wavelengths.  [a] For the determination of the quantum yield a slit width of 3.7 nm for excitation and emission monochromator path was chosen.
From this we conclude a quantum yield of about 21.4% for the partially folded form of RRB. For the lifetime determination we used a variation of the excitation and detection wavelengths to assign the different contributions to one species. From this we can assign the short lifetime of about 1.1-1.2 ns to the partially folded form, which has its emission maximum at 690 nm and the longer lifetime of about 2.0-2.2 ns to the completely folded stack, which has its emission maximum at around 790 nm.

General Procedure for the synthesis of type B building blocks (Ba-c)
Malonaldehyde dianilide hydrochloride (1.5 equiv.), the respective hydroxypyridone 1a or 1b (1.3 equiv.) and sodium acetate (1.5 eq) were suspended in acetic anhydride. The suspension was stirred at room temperature for 20 min, followed by 20 min at 90 °C. After cooling down to room temperature, the solution was dilluted with diethylether and cooled to 20 °C overnight. The formed precipitate was filtered off and the obtained solid directly mixed with the respective pyridine moiety 2a or 2b (1 equiv.) in 1,4-dioxane containing Hünig's base (1 equiv.). The mixture was heated to 100 °C and stirred at this temperature for 6 h. The reaction mixture was allowed to cool down to room temperature and then solvent was evaporated under vacuum. The obtained crude product was purified by column chromatography (eluent: CH 2 Cl 2 /MeOH = 96/4 v/v% for Ba and Bc; 94/6 v/v% for Bb) and size-exclusion chromatography (SX1, CH 2 Cl 2 /MeOH = 9:1) to yield the desired compound.

Deprotection of Ba
Monomer Ba (107 mg, 125 μmol, 1 equiv.) was dissolved in 4 mL MeOH/THF (50/50, v/v%) and 4M NaOH aqueous solution (62.5 μL, 250 μmol, 2 equiv.) was slowly added into the solution at room temperature. The mixture was stirred at room temperature for 2 h (note: the desired product slowly decomposes, therefore, the appropriate reaction time should be determined by TLC monitoring). The reaction mixture was then diluted with dichloromethane and washed with 10 mM HCl aqueous solution. The aqueous layer was extracted with 30 mL CH 2 Cl 2 three times (The addition of small amounts of MeOH might be necessary to properly dissolve the product). The organic layers were combined and (without drying over MgSO 4 or similar) condensed under vacuum. The resulting dark blue crude product was rigorously dried under high vacuum and used for the next reaction without further purification.

Deprotection of Bb
Monomer Bb (101 mg, 120 μmol, 1 equiv.) was dissolved in 6 mL dichloromethane and 4 M HCl in 1,4-dioxane (0.3 mL, 1.20 mmol, 10 equiv.) was slowly added into the dichloromethane solution at room temperature. The mixture was stirred at room temperature for 1 h (note: the desired product slowly decomposes, therefore, the appropriate reaction time should be determined by TLC monitoring; moreover, if precipitation occurs, small amounts of methanol should be added). The solvent was evaporated under vacuum as fast as possible and the residue immediately precipitated in diethyl ether to get rid of remaining HCl and solvents. The resulting dark blue crude product was rigorously dried under high vacuum and used for the next reaction without further purification.
Monomer Bc (604 mg, 999 μmol, 1 equiv.) was dissolved in 30 mL dichloromethane and 4 M HCl in 1,4-dioxane (2.5 mL, 9.99 mmol, 10 equiv.) was slowly added into the dichloromethane solution at room temperature. The mixture was stirred at room temperature for 1 h (note: the desired product slowly decomposes, therefore, the appropriate reaction time should be determined by TLC monitoring; moreover, if precipitation occurs, small amounts of methanol should be added). The solvent was evaporated under vacuum as fast as possible and the residue immediately precipitated in diethyl ether to get rid of remaining HCl and solvents. The resulting dark blue crude product was rigorously dried under high vacuum and used for the next reaction without further purification.

General Procedure for the synthesis of the intermediate dimer
The thoroughly dried acid Ba-OH or Ra-OH (1 equiv.), amine Bb-NH2 or Rb-NH2 (1.5 equiv.) and HBTU (2 equiv.) were suspended in anhydrous dichloromethane, followed by addition of Hünig's base (5 equiv.). The reaction mixture was stirred at room temperature for 1 h under nitrogen atmosphere. The solvent was evaporated under vacuum and the crude product was washed with 10 mL methanol three times. If necessary further purification was carried out by size-exclusion chromatography (SX1, CH 2 Cl 2 /MeOH = 9:1) and precipitation from methanol.