Compounds in protein

To investigate in more detail the effects of the oxyluciferin structural modification on the experimental colour modulation of the firefly’s bioluminescence, we computed the vertical energies of the S0→S1 (absorption) and S1→S0 (emission) transitions of the Natural_Oxy and its analogues at the QM/MM level considering the protein environment (Table 5) and we simulated their absorption and emission spectra. By comparing the computed results in GP and in protein, we observed that the absorption and emission transition energies computed in protein were shifted to lower energies (around 0.01–0.08 eV for the absorption and 0.05–0.2 eV for the emission) with respect to those obtained in GP (Table 5). In addition, the oscillator strengths (f) of the absorption and emission transitions computed in protein are in the same order of magnitude as those in GP, being slightly smaller when considering the protein environment than in GP. The relative order of the simulated maximum absorption and emission wavelengths in GP and in PCM are the same as the ones computed in the protein (Table 5). Moreover, the natural transition orbitals (NTOs) of the Natural_Oxy and its synthetic analogues inside the protein are quite similar to those computed in GP and in PCM (Figure A- 3).

For the purpose of determining the structural modification effect on the firefly’s bioluminescence, we compared the emission spectra of the Natural_Oxy and its analogues simulated in protein to the ones measured experimentally.^35–37 The spectral shapes of the simulated spectra are similar to the experimental ones (Figure 57). In addition, the simulated maximum emission wavelengths are only shifted by less than 10 nm with respect to the experiment. Moreover, the maximum emission wavelengths of the simulated spectra show qualitatively the same trend as the experimental ones (Figure 57). For instance, for

benzothiophene_Oxy and dihydropyrrolone_Oxy analogues, the maximum emission wavelengths are blue-shifted theoretically by 40 nm and 11 nm and experimentally by 37 nm and 13 nm, respectively, with respect to the Natural_Oxy. For the allylbenzothiazole_Oxy analogue, the simulated and experimental emission spectra are red-shifted by 46 nm and 45 nm, respectively, with respect to the Natural_Oxy. However, the simulated allylbenzothiazole_Oxy spectrum is broader than the other spectra, either theoretically or experimentally obtained (Figure 57). The origin of this spectral broadening can be due to the large fluctuation (between 40° and -160°) of the dihedral angle of the allyl group during the MD simulation (Figure A- 4e).

The simulation of 100 vertical S1→S0 (emission) transitions energy for the Natural_Oxy and its analogues shows that the optically bright transition energies are found in a narrow energy range, except for allylbenzothiazole_Oxy analogue where these transition energies are located in a larger range of energy (Figure A- 4). Hence, a Gaussian convolution of these transition energies gives a broader allylbenzothiazole_Oxy spectrum compared to the others.

Figure 57 Emission spectra of Natural_Oxy and its synthetic analogues a) simulated with QM/MM methods and b) measured experimentally.^35–37 The experimental bioluminescence spectra are adapted with permission from (C. C. Woodroofe, P. L. Meisenheimer, D. H. Klaubert, et al., Novel Heterocyclic Analogues of Firefly Luciferin, Biochemistry, 2012, 51, 9807–9813). Copyright (2012) American Chemical Society, and from (S. Ioka, T. Saitoh, S. Iwano, et al., Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties, Chem. Eur. J., 2016, 22, 9330–9337. Copyright (2016) WILEY‐VCH Verlag GmbH &

Co. KGaA, Weinheim. (Figure reproduced from ref.¹⁵¹).

It should be noted that the relative order of the simulated absorption spectra of the Natural_Oxy and its analogues in protein (Figure A- 5) is the same as the ones in the simulated and experimental emission spectra. Unfortunately, a direct comparison between the experimental and computational results cannot be made, as the experimental absorption spectra of the analogues under study are not available.

Furthermore, we investigated H-bonding between the Natural_Oxy (and its analogues) and the protein active site environment to better understand the influence of the oxyluciferin structural modifications. To determine the effect of the oxyluciferin structural modification on the H-bonding networks, we calculated a statistical average number of H-bonding between Natural_Oxy (and its analogues), water molecules, AMPH and the protein active site along the MD simulations of the system in the excited state (ES) (Figure 58a). A representative MD snapshot of each compound under study inside the protein active site showing the H-bonding is presented in Figure 58c. If we compare the H-bonding formed in the case of Natural_Oxy to that of benzothiophene_Oxy analogue, we observe that the Natural_Oxy H-bonding formed between the N2 atom (see Figure 58b for atom numbering) of the benzothiazole moiety and the SER347 and GLY246 amino acids does not exist anymore due to the structural modification (Figure 58c).

Moreover, we found that benzothiophene_Oxy analogue gets closer to AMPH structure leading to more H-bonding than in the case of the Natural_Oxy (Figure 58c and Figure A- 6a). In addition, the Natural_Oxy H-bonding formed between the oxygen atom (O10) of the phenol group and ARG337 no longer exists in the case of benzothiophene_Oxy analogue (Figure 58a and c), which can be due to the significant flexibility of the ARG amino acid side chain as already observed in previous work with Natural_Oxy.²⁸ To confirm this hypothesis, we performed a second MD simulation for the benzothiophene_Oxy analogue (Figure 59c). This second MD simulation shows that ARG337 amino acid comes close to the oxygen atom (O10) of the phenol group leading to the H-bonding. Hence, the structural modification can be excluded as reason for this finding.

Figure 58 a) Statistical average number of H-bonding involving specific atoms of the compounds under study, water molecules, AMPH and protein active site along MD simulation in the excited state (ES). b) Atom numbering. c) Representative MD snapshots of Natural_Oxy (and its analogues) inside protein active site. (Figure reproduced from ref.¹⁵¹).

Furthermore, the analysis of the MD simulations of the dihydropyrrolone_Oxy analogue and Natural_Oxy shows that the analogue structure behaves almost identically to the one of Natural_Oxy in the protein active site and it forms almost the same H-bonding, either with the surrounding water molecules and AMPH or with the amino acids (SER314, ARG218, ARG337) (Figure 58c and Figure A- 6b). Moreover, we also measured the distance between the oxygen atoms (O10 and O11, see the atom numbering in Figure 58c) of both molecules and either some amino acid (SER314, ARG218 and ARG337) atoms or the phosphate (P) of AMPH during their MD

simulations (Figure 59). We found that the measured distance between dihydropyrrolone_Oxy atoms and the amino acids or AMPH atoms are located almost at the same distances as the measured one for the Natural_Oxy (Figure 59). In addition, the calculated statistical average number of H-bonding formed during the MD simulations of both dihydropyrrolone_Oxy and Natural_Oxy are quite similar (Figure 58a). Hence, the oxyluciferin structural modification does not significantly affect the interaction between the dihydropyrrolone_Oxy structure and its surrounding. This means that the origin of the emission spectra blue shifts does not lie on different formed H-bonding.

The H-bondings formed between the allylbenzothiazole_Oxy analogue and either amino acids (SER347, ARG337, ARG218 and GLY246) or AMPH during the MD simulation are similar to those formed with the Natural_Oxy. However, the H-bond network formed between the oxygen atom (O10) of the phenol group and SER314 amino acid (close to the allyl group) no longer exists in the case of the allylbenzothiazole_Oxy analogue (Figure 58c). The measured distance between O10 atom of allylbenzothiazole_Oxy and the SER314 amino acid is around 7.5 Å, while the measured one for the Natural_Oxy is around 5.5 Å (Figure 59a). In addition, the calculated statistical average number of H-bondings formed between the O10 atom of allylbenzothiazole_Oxy analogue and the surrounding water molecules is half of that for Natural_Oxy (Figure 58a and Figure A- 7). This lack of water molecules around the oxygen atom (O10) of allylbenzothiazole_Oxy analogue can be due to the steric effect of the allyl group which moves freely (showing a large dihedral angle fluctuation between 40° and -160° in Figure A- 4e) during the MD simulation, preventing the water molecules getting closer to the oxygen atom O10.

Figure 59 a) Distance (in Å) between the oxygen atoms (O10 and O11) of the compound under study and carbon atom (CX) of amino acids (or the phosphate (P) atom of AMPH) calculated from MD simulations in the ES b) Atom numbering and the initial distances (in Å) between (O10 – CX) and (O11 – P) before the Natural_Oxy MD simulation. c) The calculated distance (in Å) between the atoms O10 and CX of ARG337 amino acid from two benzothiophene_Oxy MD simulations. (Figure reproduced from ref.¹⁵¹).

The conclusion to be drawn from this part is that the oxyluciferin structural modification slightly influences on the one hand, the distribution of the electronic density, and on the other hand, the H-bonding between the compounds and its surrounding: the protein environment, water molecules and AMPH. However, the formed H-bonding networks during MD simulations did not cause the shifts of the emission spectra, as is the case of dihydropyrrolone_Oxy spectrum blue-shift. Finally, the emission spectra of the Natural_Oxy and its analogues simulated considering the protein environment by using the QM/MM approach are in quite good agreement with the experimental bioluminescence ones.