Directing intracellular supramolecular assembly with N-heteroaromatic quaterthiophene analogues

Self-assembly in situ, where synthetic molecules are programmed to organize in a specific and complex environment i.e., within living cells, can be a unique strategy to influence cellular functions. Here we present a small series of rationally designed oligothiophene analogues that specifically target, locate and dynamically self-report their supramolecular behavior within the confinement of a cell. Through the recognition of the terminal alkyl substituent and the amphiphilic pyridine motif, we show that the cell provides different complementary pathways for self-assembly that can be traced easily with fluorescence microscopy as their molecular organization emits in distinct fluorescent bands. Importantly, the control and induction of both forms are achieved by time, temperature and the use of the intracellular transport inhibitor, bafilomycin A1. We showcase the importance of both intrinsic (cell) and extrinsic (stimulus) factors for self-organization and the potential of such a platform toward developing synthetic functional components within living cells.


Supplementary
. Normalized emission spectra of pyridine-functionalized terthiophene 1P3T (400 µM). Vibronic fine structures can be clearly seen for IsoparM in contrast to THF. Marginal shift of the maximum emission can be observed.

2-(3-(Tridecylsilyl)prop-1-yl)thiophene (1).
To a stirred suspension of magnesium turnings (11.18 g, 460 mmol) in dry THF (40 mL) a small amount of a solution of 1-bromodecane (83.0 mL, 400 mmol) in dry THF (80 mL) was added until the reaction was initiated. Subsequently, further dry THF (40 mL) and 2-(3-(trichlorosilyl)prop-1yl)thiophene (II) (14.80 g, 57 mmol) were added to the rest of the 1-bromodecane solution and this mixture was added slowly. After stirring under reflux for 19 h the reaction was quenched by addition of saturated aqueous NH 4 Cl solution and the organic phase was separated. The aqueous phase was extracted with diethyl ether and the combined organic phases were dried over MgSO 4 . Subsequently, the solvent and n-decane were removed by distillation in vacuo. Then the residual crude product was purified by column chromatography (SiO 2 /n-hexane). 1 was obtained as a colorless liquid (23.7 g, 72%). Characterization in agreement with literature. 1

Optical Spectroscopy
Fluorescence spectroscopy was performed on a Horiba FluoroMax-3 and Tecan Spark® multimode microplate reader. Absorbance measurements were conducted as well on the Tecan Spark® multimode microplate reader. All spectra were recorded at room temperature.
Oligothiophenes (4T, 1P3T, Me1P3T) were freshly dissolved in THF to afford a 1 mM stock solution. The solutions were subsequently diluted to 20 µM for absorption and fluorescence spectroscopy respectively. In co-solvent systems (i.e. THF:H 2 O = 4:1, v:v), the stock solution was always first diluted with THF followed by the addition of water to achieve the required solvent composition.
For the oligothiophene complexes (4T-HSA, 1P3T-HSA, Me1P3T-HSA), the solutions were first dissolved into a stock solution of 300 µM using MilliQ water. The samples were further diluted into triplicates to afford 20 µM. The absorbance and fluorescence were then measured in a 384-well UVstar® microplate (Greiner Bio-One).
To achieve steady-state aggregation/disaggregation of the respective oligothiophenes, all prepared solutions were left standing at room temperature for 24 h before measurement. Each solution was excited at 400 nm with emission scan from 420 -750 nm.

Fluorescence Correlation Spectroscopy (FCS)
FCS experiments were performed on a commercial confocal microscope, LSM 880 (Carl Zeiss, Jena, Germany) equipped with a C-Apochromat 40x/1.2W water immersion objective. The fluorophores were excited by a diode laser (405 nm) fiber-coupled to the microscope. Emitted fluorescence light was collected with the same objective, passed through a confocal pinhole and directed to a spectral detection unit (Quasar, Carl Zeiss). In this unit emission is spectrally separated by a grating element on a 32 channel array of GaAsP detectors operating in a single photon counting mode. In all experiments the emission in the spectral range 420 -700 nm was considered. A stainless steel chamber Attofluor (Thermo Fisher Scientific) holding a 25 mm round coverslip was used as a sample cell for the studied solutions. For each sample, a series of measurements with a total duration of 5 min were performed. The time-dependent fluctuations of the fluorescent intensity I(t) were recorded and analyzed by an autocorrelation function G()=1+<I(t)I(t+)>/<I(t)> 2 . As it has been shown theoretically for an ensemble of m different types of freely diffusing identical fluorescence species, G() has the following analytical form 6 : Here, N is the average number of diffusing fluorescence species in the observation volume, f T and  T are the fraction and the decay time of the triplet state, S = z 0 /r 0 is the so-called structure parameter with z 0 and r 0 representing the axial and radial dimensions of the confocal volume. F i is the fraction of the i-th species and  Di is their diffusion time through the observation volume that is related to their diffusion coefficient, D i , through D i = r 0 2 /4 Di. The experimentally obtained G() were fitted with Supplementary Equation 1, yielding the corresponding diffusion times and subsequently the diffusion coefficients of the fluorescent species. Finally, the hydrodynamic radii R h were calculated (assuming spherical particles) using the Stokes-Einstein relation: R h = k B T/6D, where k B is the Boltzmann constant, T is the temperature, and  is the viscosity of the solution. As the value of r 0 depends strongly on the specific characteristics of the optical setup a calibration was done using a reference standard with known diffusion coefficient, i.e. ATTO 425.