Stereoselectivity of Electron and Energy Transfer in the Quenching of (S/R)-Ketoprofen-(S)-Tryptophan Dyad Excited State

Photoinduced elementary processes in chiral linked systems, consisting of drugs and tryptophan (Trp) residues, attract considerable attention due to several aspects. First of all, these are models that allow one to trace the full and partial charge transfer underlying the binding of drugs to enzymes and receptors. On the other hand, Trp fluorescence is widely used to establish the structure and conformational mobility of proteins due to its high sensitivity to the microenvironment. Therefore, the study of mechanisms of Trp fluorescence quenching in various systems has both fundamental and practical interest. An analysis of the photo-chemically induced dynamic nuclear polarization (CIDNP) and Trp fluorescence quenching in (R/S)-ketoprofen-(S)-tryptophan ((S/R)-KP-(S)-Trp) dyad carried out in this work allowed us to trace the intramolecular reversible electron transfer (ET) and obtain evidence in favor of the resonance energy transfer (RET). The fraction of dyad’s singlet excited state, quenched via ET, was shown to be 7.5 times greater for the (S,S)-diastereomer than for the (R,S) analog. At the same time, the ratio of the fluorescence quantum yields shows that quenching effectiveness of (S,S)-diastereomer to be 5.4 times lower than for the (R,S) analog. It means that the main mechanism of Trp fluorescence quenching in (S/R)-KP-(S)-Trp dyad is RET.


Synthesis of (S/R)-ketoprofen-(S)-tryptophan dyad
The synthesis was carried out from ketoprofen and tryptophan according to the Materials. All reagents were ACS grade, and were used without further purification.
The UV-254 plates were used for TLC analysis. The IR-spectra were recorded in KBr pellets. Mass spectra (HRMS) were measured on at 70 eV. NMR spectra were recorded at 500 ( 1 H) at 25 °C. Chemical shifts (δ) are given in ppm with reference to the residual signals of [D1] chloroform (1H: δ = 7.24 ppm). The mass spectra (High Resolution GC/MS) were measured by the direct injection method (the temperature of the ionization chamber was 220−270 °C and the ionization voltage was 70 eV).
The analysis of the dyads' purity was carried out by thin layer chromatography (TLC), IR, high resolution GC / MS and NMR techniques. Dyads' purity studies did not reveal the presence of Trp impurities. The possibility of its formation during the photodecomposition of the dyad was excluded by monitoring this process by high resolution NMR.   Figure S1) of the both isomers of dyad (since they are strongly spaced in the spectra ( Figures S2-S3)) were exposed to RF irradiation. These are intense emission lines in the spectra. All absorption lines are those protons that are located next to the irradiated protons (as a rule, protons located at a distance of up to 4.5 Å are visible). The number of scans and concentration are the same. When 23 proton of (R,S) dyad is irradiated, a cross peak is observed at the ortho/para protons of KP (marked with an oval), while when the same proton of (S,S) dyad is excited, there is no such peak. It let us to suggest that tryptophan and ketoprofen rings in (R,S) dyad are closer to each other than in (S,S) analog. Figure S4. 1D NOE spectra of (R,S) (green) and (S,S) (blue) diastereomers. When 23 proton of tryptophan fragment of (R,S) dyad is excited, a cross peak is observed at the ortho/para protons of KP (marked with an oval), while when this proton of (S,S) dyad is excited, there is no such peak.

CIDNP analysis
An analysis of CIDNP spectra detected at high magnetic field can be obtained by wellknown Kaptein's rules, modified by Closs [3], taking into account the possibility of biradical-zwitterion recombination from both spin states. For the net effect, the sign can be determined by the following parameters where µ denotes the initial spin multiplicity (+ for triplet, − for singlet), ε the type of reaction leading to the observed products (+ for cage product, − for escaped products), Δg = g1−g2 the sign of difference in g-factors of the two radicals (g1 is the gfactor of the radical ion with nucleus under observation), ai the sign of the HFI constant and 'exit channel' factor γ (+ for singlet exit channel, -for triplet). The sign product of these parameters determines whether the sign, Гi, of the polarization for nucleus i indicates absorption (A) or emission (E). and S-Tryptophan methyl ester in solution are shown in Figure S5.

Quantum chemical calculation of various configurations of diastereomers of a dyad (S/R) ketoprofen-(S) tryptophan
Calculations were performed using software Hyperchem 8 by semi-empirical AM1 method. Energy of dyads spatial configuration was minimized with restriction of selected torsion angle and optimization of all other geometrical parameters.
The pictures presented below show the results of calculations of the dependence of the values of the heat of formation, the distances between donor and acceptor of dyad's diastereomers, and the angle between the planes of the ketoprofen and tryptophan fragments upon rotation of one bond (Figures S8-S10). This is specifically the distances between carbonyl carbon of KP and nitrogen atom of indole fragment of dyad (intercenter distance) and the angles between planes of Trp aromatic ring and "C-CO-C" fragment of KP (interplane angle) calculated for different torsion angle. The rotation was carried out about С -С bond of molecular bridge shown in the Figure S8   In general, it is seen that for (R,S)-diastereomer, the rotation about bonds of bridge leads to significantly bigger removal of fragments from each other than for (S,S). It can be assumed that it is due to the arrangement of bulky substituents relative to chiral centers, where they can more or less interfere with each other under rotation ( Figure   S11).