Topological effects in ultrafast photoinduced processes between flurbiprofen and tryptophan in linked dyads and within human serum albumin

The interaction dynamics between flurbiprofen (FBP) and tryptophan (Trp) has been studied in covalently linked dyads and within human serum albumin (HSA) by means of fluorescence and ultrafast transient absorption spectroscopy. The dyads have proven to be excellent models to investigate photoinduced processes such as energy and/or electron transfer that may occur in proteins and other biological media. Since the relative spatial arrangement of the interacting units may affect the yield and kinetics of the photoinduced processes, two spacers consisting of amino and carboxylic groups separated by a cyclic or a long linear hydrocarbon chain (1 and 2, respectively) have been used to link the (S)- or (R)-FBP with the (S)-Trp moieties. The main feature observed in the dyads was a strong intramolecular quenching of the fluorescence, which was more important for the (S,S)- than for the (R,S)- diastereomer in dyads 1, whereas the reverse was true for dyads 2. This was consistent with the results obtained by simple molecular modelling (PM3). The observed stereodifferentiation in (S,S)-1 and (R,S)-1 arises from the deactivation of 1Trp*, while in (S,S)-2 and (R,S)-2 it is associated with 1FBP*. The mechanistic nature of 1FBP* quenching is ascribed to energy transfer, while for 1Trp* it is attributed to electron transfer and/or exciplex formation. These results are consistent with those obtained by ultrafast transient absorption spectroscopy, where 1FBP* was detected as a band with a maximum at ca. 425 nm and a shoulder at ∼375 nm, whereas Trp did not give rise to any noticeable transient. Interestingly, similar photoprocesses were observed in the dyads and in the supramolecular FBP@HSA complexes. Overall, these results may aid to gain a deeper understanding of the photoinduced processes occurring in protein-bound drugs, which may shed light on the mechanistic pathways involved in photobiological damage.

and (R,S)-2 (dark blue) after excitation at 250 nm in acetonitrile. The simulated spectrum that is obtained considering the percentage of photons absorbed by isolated FBP (90%) and TrpMe (10%) at 250 nm and assuming no interactions between them in their excited states is shown in violet.     Tetrahydrofuran (THF), pyridine, methylene chloride, ethyl acetate and acetonitrile (HPLC quality) were from Scharlab. PBS buffer was prepared by dissolving phosphatebuffered saline tablets (Sigma) using ultrapure water from a Millipore (Milli-Q Synthesis) system.
A semipreparative JASCO HPLC system (PU-2080 Plus pump, DG-2080-54-line degasser and LG-2080-04 gradient unit) connected to a JASCO (UV-1575) detector was used to further purify the different products, using an isocratic flux (2 mL/min) of the appropriate organic solvent as an eluent, and a SEA18 Teknokroma column or a Tecknokroma TR-016178 NF-33978 Tracer Excel 120 31 column, 5 μm (25 × 1 cm 2 ). Spectroscopic Techniques. The 1 H-and 13 C-NMR spectra were recorded at 400 and 100 MHz, respectively, using a Bruker AVANCE III instrument; chemical shifts are reported in ppm.

High-resolution mass spectrometry (HRMS) was performed in an Ultra Performance
Liquid Chromatography (UPLC) ACQUITY system (Waters Corp.) with a conditioned autosampler at 4 °C. The separation was accomplished on an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm), which was maintained at 40 °C. The analysis was performed using acetonitrile and water (60:40 v/v containing 0.01% formic acid) as the mobile phase with a flow rate of 0.5 mL/min, and injection volume was 5 μL. The Waters S5 ACQUITY™ XevoQToF Spectrometer (Waters Corp.) was connected to the UPLC system via an electrospray ionization (ESI) interface. This source was operated in positive ionization mode with the capillary voltage at 1.5 kV at 100 °C and the temperature of the desolvation was 300 °C. The cone and desolvation gas flows were 40 L h −1 and 800 L h −1 , respectively. The collision gas flow and collision energy applied were 0.2 mL/min and 12 V, respectively. All data collected in Centroid mode were acquired using Masslynx™ software (Waters Corp.). Leucine-enkephalin was used at a concentration of 500 pg/L as the lock mass generating an [M + H] + ion (m/z 556.2771) and fragment at m/z 120.0813 and flow rate of 50 μL/min to ensure accuracy during the MS analysis.
Steady-state absorption spectra were recorded in a JASCO V-760 spectrophotometer.
Steady-state fluorescence measurements (exc = 266 nm) were performed on an Edinburgh FS5 spectrofluorometer, provided with a monochromator in the wavelength range of 200-900 nm. Time-resolved fluorescence measurements were done using an EasyLife X system containing a sample compartment composed of an automated Peltier cuvette holder to control the temperature at 24 ºC, a pulsed LED excitation source and a lifetime detector. The employed LED excitation source was 265 nm, with emission filter of WG305 and/or WG420. The absorbance of the samples was identical (ca. 0.1) at the excitation wavelength.
Laser Flash Photolysis (LFP) measurements were performed using a pulsed Nd:YAG L52137 V LOTIS TII at exc = 266 nm. The single pulses were ca. 10 ns duration, and the energy was ~12 mJ/pulse. The laser flash photolysis system consisted of the pulsed laser, a 77250 Oriel monochromator and an oscilloscope DP04054 Tektronix. The output signal from the oscilloscope was transferred to a personal computer. Absorbances of all solutions were adjusted at ~0.20 at 266 nm in acetonitrile (HPLC grade). All UV, S6 fluorescence and LFP measurements were done using 10 × 10 mm 2 quartz cuvettes at room temperature in deaerated acetonitrile (25 min N2 bubbling). Control experiments indicated that the degree of decomposition of the samples after photolysis was lower than 5%.
Femtosecond transient absorption experiments were performed using a pump-probe system. The femtosecond pulses were generated with a mode-locked Ti-Sapphire laser of a compact Libra HE (4 W power at 4 kHz) regenerative amplifier delivering 100 fs pulses at 800 nm (1 mJ/pulse). The output of the laser was split into two parts to generate the pump and the probe beams. Thus, tunable femtosecond pump pulses were obtained by directing the 800 nm light into an optical parametric amplifier (OPA). In the present case, the 800 nm beam was transformed into the 250 nm exciting beam after performing proper alignment of the BBO crystals, optimizing the values of the two delay lines and using the appropriate mixers within the OPA. The 250 nm pump beam passed through a chopper prior to focus onto a rotating cell (1 mm optical path) containing the sample. The white light used as probe was produced after part of the 800 nm light from the amplifier travelled through a computer controlled 8 ns variable optical delay line and impinge on a CaF2 rotating crystal. This white light was in turn split in two identical portions to generate reference and probe beams that then were focused on the rotating cell containing the sample. The pump and the probe beams were made to coincide to interrogate the sample.
The power of the pump beam was set to 180 µW. Under these conditions, the degree of photodegradation of the samples was lower than 5%. A computer-controlled imaging spectrometer was placed after this path to measure the probe and the reference pulses to obtain the transient absorption decays/spectra. The experimental data were treated and compensated by the chirp using the ExciPro program. The data files were exported as matrix format from ExciPro to be treated with OriginLab program to get the time-resolved S7 spectra and the ultrafast decay traces, which were fitted using a multi-exponential function following the Levenberg-Marquardt iteration algorithm: ( , ) = ∑ ( )ⅇ (− ∕ ) =1 + 0 with n = 2 or 3.