# Assessing Configurational Sampling in the Quantum Mechanics/Molecular Mechanics Calculation of Temoporfin Absorption Spectrum and Triplet Density of States

^{1}

^{2}

^{3}

^{*}

^{†}

## Abstract

**:**

## 1. Introduction

^{®}[18,19]. mTHPC is currently employed in clinics [1,2,3,13,19,20,21] and it is being further developed into an assisted delivery formulation, where the drug is embedded in liposomes [22]. This new formulation should expand the applications of the drug and improve its efficacy [23,24,25,26].

## 2. Results and Discussion

#### 2.1. Absorption Spectrum

_{b}T provided by the sampling approaches based on classical or QM/MM MD. A configurational space generated with quantum sampling is therefore generally hotter than the correspondent ensemble generated with a thermal sampling. Only the low-frequency modes could receive the same amount of energy in both sampling approaches and those are also the modes that could deviate most from the harmonic approximation. As the 100 geometries obtained with the quantum sampling are strictly harmonic, the comparison with the QM/MM-MD approach allows determining whether the improvement in the QM/MM-MD-based absorption spectrum with respect to the classical MD-based one is due to a better description of the anharmonic motions or not. In order to keep the comparison rigorously bound to the harmonic character of the configurations of the chromophore in the ensemble, the solvent has to be described in the same fashion as in the QM/MM-MD ensemble. This was achieved by taking the previously selected 100 snapshots of the classical MD simulation and replacing the geometries of the chromophore by the geometries from the Wigner distribution. Such snapshots were then the starting point for 100 classical simulations of 1 ns, where the geometry of the drug was kept frozen and the solvent relaxes to adapt to the new structure of the chromophore. The hybrid-Wigner-MD ensemble formed by the last snapshot from each of these 100 classical MD simulations was employed for excited-state calculations.

#### 2.2. Density of Triplet States

^{1}O

_{2}, fundamental for the therapeutic effect, are produced from the reaction of the PS in a triplet electronic excited state with compounds of the surrounding environment; this can be molecular oxygen or a more complex molecular substrate, such as the cell membrane. It is therefore interesting to compute the density of triplet states and to investigate how different computation protocols affect their energies.

_{1}band presents a portion of its DOS close and below 0 eV. When the sampling is performed by classical MD, there is also a portion of the triplet band falling below zero. The problem of having negative triplet transition energies in TD-DFT calculations [36,48,50] can be partially ascribed to the exchange-correlation functional, where the amount of Hartree-Fock exchange [51] and the range-separated character [49] are the most delicate aspects in this matter. A second source of error in TD-DFT is the adiabatic approximation which assumes an instantaneous reaction of the exchange-correlation potential when the electron density changes in time. It has been shown that this approximation introduces large errors when computing spin-flip transitions, such as S

_{0}-T

_{1}transitions, especially when range-separated functionals, for example, wB97XD, are used [49]. Exchange and correlational functionals that present relatively large contributions of the HF exchange have larger probability to display the so-called triplet instability problem, for which the triplet excitation energies approach the zero value [51].

_{0}-T

_{1}energies, we have found that the use of different functionals for sampling (B3LYP) and excited-state energies (wB97XD) also introduces a significant error. Indeed, if the same functional is used (wB97XD) for both types of calculations the negative energy problem vanishes, as can be seen in Figure 5b. The DOS of the first triplet excited state is now centred at 0.93 eV (i.e., blue-shifted by 0.17 eV with respect to using B3LYP) and there are no geometries with negative triplet states. Figure 5 also shows that the use of different functionals for sampling does not significantly affect the excitation energies of singlet excited states since the calculated absorption spectra based on QM(B3LYP)/MM-MD and QM(wB97XD)/MM-MD sampling protocols are similar, with a small blue shift of 0.07 eV of the latter with respect to the former. Therefore, the spectral bands computed with QM(wB97XD)/MM show a larger blue-shift for the triplets (0.17 eV) than for the singlets (0.07 eV) when the sampling is changed from QM(B3LYP)/MM-MD to QM(wB97XD)/MM-MD. In other words, the use of a different functional for sampling and excitation energies calculation introduces an artificial red-shift of the excitation energies, which is more important for the triplet states and thus causes the appearance of negative triplet-state energies for some geometries of the ensemble.

_{n}excitation energies are only shifted by a maximum of 0.21 eV to lower energies with respect to the values obtained using the wB97XD functional for both geometry optimization and excited-state calculations. Triplet electronic transitions T

_{n}, in contrast, suffer from stronger deviations and show red-shifts of even 0.45 eV when a different functional is adopted for the geometry optimization and the excited-state calculation.

## 3. Conclusions

_{1}states.

## 4. Materials and Methods

#### 4.1. Computational Details

^{−1}while the pressure was kept fixed at 1 atm with the Berendsen barostat and isotropic position scaling. The cut-off for the non-bonded interactions was set to 10.0 Å and the particle-mesh Ewald method was used to calculate the coulombic interactions [57].

_{opt}= 1.77.

#### 4.2. Experimental Methods

^{®}quartz cuvettes (HELLMA, Müllheim, Germany). Temoporfin (mTHPC) was taken from a lot delivered by biolitec pharma Ltd. (Dublin, Ireland). The methanol solvent used to prepare the Temoporfin solution was of spectroscopic grade (MERCK, Uvasol

^{®}). The obtained experimental spectrum of Temoporfin in methanol was normalized to 1 at the highest absorption peak in the UV/vis region and agrees with the peaks reported in Ref. [17,18,61].

## Supplementary Materials

## Author Contributions

## Funding

## Acknowledgments

## Conflicts of Interest

## References

- Copper, M.P.; Tan, I.B.; Oppelaar, H.; Ruevekamp, M.C.; Stewart, F.A. Meta-tetra(hydroxyphenyl)chlorin Photodynamic Therapy in Early-Stage Squamous Cell Carcinoma of the Head and Neck. Arch. Otolaryngol.-Head Neck Surg.
**2003**, 129, 709–711. [Google Scholar] [CrossRef] [PubMed] - Fan, K.F.; Hopper, C.; Speight, P.M.; Buonaccorsi, G.A.; Bown, S.G. Photodynamic therapy using mTHPC for malignant disease in the oral cavity. Int. J. Cancer
**1997**, 73, 25–32. [Google Scholar] [CrossRef] [Green Version] - Agostinis, P.; Berg, K.; Cengel, K.; Foster, T.; Girotti, A.; Gollnick, S.; Hahn, S.; Hamblin, M.; Juzeniene, A.; Kessel, D.; et al. Photodynamic Terapy of cancer: An update. CA Cancer J. Clin.
**2011**, 61, 250–281. [Google Scholar] [CrossRef] [PubMed] - Dąbrowski, J.M.; Arnaut, L.G. Photodynamic therapy (PDT) of cancer: From local to systemic treatment. Photochem. Photobiol. Sci.
**2015**, 14, 1765–1780. [Google Scholar] [CrossRef] [PubMed] - Hamblin, M.R.; Hasan, T. Photodynamic therapy: A new antimicrobial approach to infectious disease? Photochem. Photobiol. Sci.
**2004**, 3, 436–450. [Google Scholar] [CrossRef] [PubMed] - Tardivo, J.P.; Adami, F.; Correa, J.A.; Pinhal, M.A.S.; Baptista, M.S. A clinical trial testing the efficacy of PDT in preventing amputation in diabetic patients. Photodiagn. Photodyn. Ther.
**2014**, 11, 342–350. [Google Scholar] [CrossRef] [PubMed] - Goldberg, D.J. Photodynamic therapy in skin rejuvenation. Clin. Dermatol.
**2008**, 26, 608–613. [Google Scholar] [CrossRef] [PubMed] - Lee, Y.; Baron, E.D. Photodynamic therapy: Current evidence and applications in dermatology. Semin. Cutan. Med. Surg.
**2011**, 30, 199–209. [Google Scholar] [CrossRef] [PubMed] - Zhu, T.C.; Finlay, J.C. The role of photodynamic therapy (PDT) physics. Med. Phys.
**2008**, 35, 3127–3136. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Bacellar, I.O.L.; Tsubone, T.M.; Pavani, C.; Baptista, M.S. Photodynamic Efficiency: From Molecular Photochemistry to Cell Death. Int. J. Mol. Sci.
**2015**, 16, 20523–20559. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R.K. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem. Soc. Rev.
**2011**, 40, 340–362. [Google Scholar] [CrossRef] [PubMed] - Josefsen, L.B.; Boyle, R.W. Unique diagnostic and therapeutic roles of porphyrins and phthalocyanines in photodynamic therapy, imaging and theranostics. Theranostics
**2012**, 2, 916–966. [Google Scholar] [CrossRef] [PubMed] - Bown, S.G.; Rogowska, A.Z. New photosensitizers for photodynamic therapy in gastroenterology. Can. J. Gastroenterol.
**1999**, 13, 389–392. [Google Scholar] [CrossRef] [PubMed] - Bonnett, R.; White, R.D.; Winfield, U.J.; Berenbaum, M.C. Hydroporphyrins of the meso-tetra(hydroxyphenyl)porphyrin series as tumour photosensitizers. Biochem. J.
**1989**, 261, 277–280. [Google Scholar] [CrossRef] [PubMed] - Mehraban, N.; Freeman, H. Developments in PDT Sensitizers for Increased Selectivity and Singlet Oxygen Production. Materials
**2015**, 8, 4421–4456. [Google Scholar] [CrossRef] [PubMed] [Green Version] - DeRosa, M.C.; Crutchley, R.J. Photosensitized singlet oxygen and its applications. Coord. Chem. Rev.
**2002**, 233–234, 351–371. [Google Scholar] [CrossRef] - Palma, M.; Cardenas-Jiron, G.I.; Rodriguez, M.I.M. Effect of chlorin structure on theoretical electronic absorption spectra and on the energy released by porphyrin-based photosensitizers. J. Phys. Chem. A
**2008**, 112, 13574–13583. [Google Scholar] [CrossRef] [PubMed] - Bonnett, R.; Djelal, B.D.; Nguyen, A. Physical and chemical studies related to the development of m-THPC (FOSCAN (R)) for the photodynamic therapy (PDT) of tumours. J. Porphyr. Phthalocyanines
**2001**, 5, 652–661. [Google Scholar] [CrossRef] - Senge, M.O.; Brandt, J.C. Temoporfin (Foscan
^{®}, 5,10,15,20-Tetra(m-hydroxyphenyl)chlorin)—A second-generation photosensitizer. Photochem. Photobiol.**2011**, 87, 1240–1296. [Google Scholar] [CrossRef] [PubMed] - Betz, C.S.; Rauschning, W.; Stranadko, E.P.; Riabov, M.V.; Volgin, V.N.; Albrecht, V.; Nifantiev, N.E.; Hopper, C. Long-term outcomes following Foscan
^{®}-PDT of basal cell carcinomas. Lasers Surg. Med.**2012**, 44, 533–540. [Google Scholar] [CrossRef] [PubMed] - Huang, Z. A review of progress in clinical photodynamic therapy. Technol. Cancer Res. Treat.
**2005**, 4, 283–293. [Google Scholar] [CrossRef] [PubMed] - De Vetta, M.; González, L.; Nogueira, J.J. Hydrogen Bonding Regulates the Rigidity of Liposome-Encapsulated Chlorin Photosensitizers. ChemistryOpen
**2018**, 7, 475–483. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Senge, M.O. mTHPC—A drug on its way from second to third generation photosensitizer? Photodiagn. Photodyn. Ther.
**2012**, 9, 170–179. [Google Scholar] [CrossRef] [PubMed] - Meier, D.; Botter, S.M.; Campanile, C.; Robl, B.; Gräfe, S.; Pellegrini, G.; Born, W.; Fuchs, B. Foscan and foslip based photodynamic therapy in osteosarcoma in vitro and in intratibial mouse models. Int. J. Cancer
**2017**, 140, 1680–1692. [Google Scholar] [CrossRef] [PubMed] - Reshetov, V.; Kachatkou, D.; Shmigol, T.; Zorin, V.; D’Hallewin, M.-A.; Guillemin, F.; Bezdetnaya, L. Redistribution of meta-tetra(hydroxyphenyl)chlorin (m-THPC) from conventional and PEGylated liposomes to biological substrates. Photochem. Photobiol. Sci.
**2011**, 10, 911–919. [Google Scholar] [CrossRef] [PubMed] - Pegaz, B.; Debefve, E.; Ballini, J.P.; Wagnières, G.; Spaniol, S.; Albrecht, V.; Scheglmann, D.V.; Nifantiev, N.E.; Van Den Bergh, H.; Konan-Kouakou, Y.N. Photothrombic activity of m-THPC-loaded liposomal formulations: Pre-clinical assessment on chick chorioallantoic membrane model. Eur. J. Pharm. Sci.
**2006**, 28, 134–140. [Google Scholar] [CrossRef] [PubMed] - Mazzone, G.; Alberto, M.E.; De Simone, B.C.; Marino, T.; Russo, N. Can expanded bacteriochlorins act as photosensitizers in photodynamic therapy? Good news from density functional theory computations. Molecules
**2016**, 21, 288. [Google Scholar] [CrossRef] [PubMed] - Eriksson, E.S.E.; Eriksson, L.A. Predictive power of long-range corrected functionals on the spectroscopic properties of tetrapyrrole derivatives for photodynamic therapy. Phys. Chem. Chem. Phys.
**2011**, 13, 7207–7217. [Google Scholar] [CrossRef] [PubMed] - Alberto, M.E.; Marino, T.; Quartarolo, A.D.; Russo, N. Photophysical origin of the reduced photodynamic therapy activity of temocene compared to Foscan
^{®}: Insights from theory. Phys. Chem. Chem. Phys.**2013**, 15, 16167–16171. [Google Scholar] [CrossRef] [PubMed] - Cossi, M.; Barone, V. Solvent effect on vertical electronic transitions by the polarizable continuum model. J. Chem. Phys.
**2000**, 112, 2427. [Google Scholar] [CrossRef] - Mennucci, B. Polarizable continuum model. Wiley Interdiscip. Rev. Comput. Mol. Sci.
**2012**, 2, 386–404. [Google Scholar] [CrossRef] - De Vetta, M.; Menger, M.F.S.J.; Nogueira, J.J.; González, L. Solvent Effects on Electronically Excited States: QM/Continuum Versus QM/Explicit Models. J. Phys. Chem. B
**2018**, 122, 2975–2984. [Google Scholar] [CrossRef] [PubMed] - Senn, H.M.; Thiel, W. QM/MM methods for biomolecular systems. Angew. Chem. Int. Ed.
**2009**, 48, 1198–1229. [Google Scholar] [CrossRef] [PubMed] - Van Der Kamp, M.W.; Mulholland, A.J. Combined quantum mechanics/molecular mechanics (QM/MM) methods in computational enzymology. Biochemistry
**2013**, 52, 2708–2728. [Google Scholar] [CrossRef] [PubMed] - Nogueira, J.J.; Roßbach, S.; Ochsenfeld, C.; González, L. Effect of DNA Environment on Electronically Excited States of Methylene Blue Evaluated by a Three-Layered QM/QM/MM ONIOM Scheme. J. Chem. Theory Comput.
**2018**, 14, 4298–4308. [Google Scholar] [CrossRef] [PubMed] - Adamo, C.; Jacquemin, D. The calculations of excited-state properties with Time-Dependent Density Functional Theory. Chem. Soc. Rev.
**2013**, 42, 845–856. [Google Scholar] [CrossRef] [PubMed] - Zuehlsdorff, T.J.; Isborn, C.M. Combining the ensemble and Franck-Condon approaches for calculating spectral shapes of molecules in solution. J. Chem. Phys.
**2018**, 148. [Google Scholar] [CrossRef] [PubMed] - Milanese, J.M.; Provorse, M.R.; Alameda, E.; Isborn, C.M. Convergence of Computed Aqueous Absorption Spectra with Explicit Quantum Mechanical Solvent. J. Chem. Theory Comput.
**2017**, 13, 2159–2171. [Google Scholar] [CrossRef] [PubMed] - Nåbo, L.J.; Olsen, J.M.H.; Martínez, T.J.; Kongsted, J. The Quality of the Embedding Potential Is Decisive for Minimal Quantum Region Size in Embedding Calculations: The Case of the Green Fluorescent Protein. J. Chem. Theory Comput.
**2017**, 13, 6230–6236. [Google Scholar] [CrossRef] [PubMed] - Nogueira, J.J.; González, L. Computational Photophysics in the Presence of an Environment. Annu. Rev. Phys. Chem.
**2018**, 69, 473–497. [Google Scholar] [CrossRef] [PubMed] - Zobel, J.P.; Nogueira, J.J.; González, L. Finite-temperature Wigner phase-space sampling and temperature effects on the excited-state dynamics of 2-nitronaphthalene. Phys. Chem. Chem. Phys.
**2018**. [Google Scholar] [CrossRef] [PubMed] - Plasser, F.; Aquino, A.J.A.; Hase, W.L.; Lischka, H. UV absorption spectrum of alternating DNA duplexes. Analysis of excitonic and charge transfer interactions. J. Phys. Chem. A
**2012**, 116, 11151–11160. [Google Scholar] [CrossRef] [PubMed] - Nogueira, J.J.; Plasser, F.; González, L. Electronic delocalization, charge transfer and hypochromism in the UV absorption spectrum of polyadenine unravelled by multiscale computations and quantitative wavefunction analysis. Chem. Sci.
**2017**, 8, 5682–5691. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Parac, M.; Doerr, M.; Marian, C.M.; Thiel, W. QM/MM Calculation of Solvent Effects on Absorption Spectra of Guanin. J. Comput. Chem.
**2009**, 30, 1545–1614. [Google Scholar] [CrossRef] - Etienne, T.; Assfeld, X.; Monari, A. QM/MM calculation of absorption spectra of complex systems: The case of human serum albumin. Comput. Theor. Chem.
**2014**, 1040–1041, 360–366. [Google Scholar] [CrossRef] - Kjellgren, E.R.; Haugaard Olsen, J.M.; Kongsted, J. Importance of Accurate Structures for Quantum Chemistry Embedding Methods: Which Strategy Is Better? J. Chem. Theory Comput.
**2018**, 14, 4309–4319. [Google Scholar] [CrossRef] [PubMed] - González, L.; Escudero, D.; Serrano-Andrés, L. Progress and challenges in the calculation of electronic excited states. ChemPhysChem
**2012**, 13, 28–51. [Google Scholar] [CrossRef] [PubMed] - Serrano-Andrés, L.; Merchán, M. Quantum chemistry of the excited state: 2005 overview. J. Mol. Struct. THEOCHEM
**2005**, 729, 99–108. [Google Scholar] [CrossRef] - Cui, G.; Yang, W. Challenges with range-separated exchange-correlation functionals in time-dependent density functional theory calculations. Mol. Phys.
**2010**, 108, 2745–2750. [Google Scholar] [CrossRef] - Cai, Z.-L.; Sendt, K.; Reimers, J.R. Failure of density-functional theory and time-dependent density-functional theory for large extended π systems. J. Chem. Phys.
**2002**, 117, 5543–5549. [Google Scholar] [CrossRef] - Peach, M.J.G.; Tozer, D.J. Overcoming low orbital overlap and triplet instability problems in TDDFT. J. Phys. Chem. A
**2012**, 116, 9783–9789. [Google Scholar] [CrossRef] [PubMed] - Wang, J.M.; Wolf, R.M.; Caldwell, J.W.; Kollman, P.A.; Case, D.A. Development and testing of a general amber force field. J. Comput. Chem.
**2004**, 25, 1157–1174. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Becke, A.D. A new mixing of Hartree-Fock and local density-functional theories. J. Chem. Phys.
**1993**, 98, 1372–1377. [Google Scholar] [CrossRef] - Ditchfield, R.; Hehre, W.J.; Pople, J.A. An Extended Gaussian-Type Basis for Molecular-Orbital Studies of Organic Molecules. J. Chem. Phys.
**1971**, 54, 724–728. [Google Scholar] [CrossRef] - Cieplak, P.; Caldwell, J.; Kollman, P. Molecular mechanical models for organic and biological systems going beyond the atom centered two body additive approximation: Aqueous solution free energies of methanol and N-methyl acetamide, nucleic acid base, and amide hydrogen bonding and chloroform/water partition coefficients of the nucleic acid bases. J. Comput. Chem.
**2001**, 22, 1048–1057. [Google Scholar] [CrossRef] - Miyamoto, S.; Kollman, P. Settle, A. An analytical version of the SHAKE and RATTLE algorithm for rigid water models. J. Comput. Chem.
**1992**, 13, 952–962. [Google Scholar] [CrossRef] - Crowley, M.F.; Darden, T.A.; Cheatham, T., III; Deerfield, D., II. Adventures in improving the scaling and accuracy of a parallel molecular dynamics program. J. Supercomput.
**1997**, 11, 255–278. [Google Scholar] [CrossRef] - Chai, J.-D.; Head-Gordon, M. Long-range corrected hybrid density functionals with damped atom–atom dispersion corrections. Phys. Chem. Chem. Phys.
**2008**, 10, 6615. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Case, D.A.; Cerutti, D.S.; Cheatham, T.E., III; Darden, T.A.; Duke, R.E.; Giese, T.J.; Gohlke, A.W.; Goetz, D.G.; Homeyer, S.I.; Kovalenko, A.; et al. AMBER16; University of California: San Francisco, CA, USA, 2017. [Google Scholar]
- Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Petersson, G.A.; Nakatsuji, H.; et al. Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford, CT, USA, 2009. [Google Scholar]
- Bonnett, R.; Charlesworth, P.; Djelal, B.D.; Foley, S.; McGarvey, D.J.; Truscott, T.G. Photophysical properties of 5,10,15,20-tetrakis(m-hydroxyphenyl)-porphyrin (m-THPP), 5,10,15,20-tetrakis (m-hydroxyphenyl) chlorin (m-THPC) and 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (m-THPBC): A comparative study. J. Chem. Soc. Perkin Trans.
**1999**, 2, 325–328. [Google Scholar] [CrossRef]

Sample Availability: Samples of the compound mTHPC are available upon request. |

**Scheme 1.**Structural representation of the second-generation photosensitizer mTHPC (5,10,15,20-tetra(m-hydroxyphenyl)chlorin).

**Figure 1.**Comparison of the QM(wB97XD)/MM@Classical-MD spectrum (red curve) and the experimental spectrum (black), scaled with respect to the Soret band. In the inset a zoom on the Q-bands with the corresponding energy values at the maxima in eV is reported.

**Figure 2.**Comparison of the @100-QM(B3LYP)/MM-MD spectrum (blue) with respect to the experimental absorption curve of mTHPC in methanol (black) and the one obtained with the classical MD sampling (red). The spectra are scaled with respect to the Soret band. The inset zooms the Q’ and Q′′ bands. Values of the three peaks of the @100-QM(B3LYP)/MM-MD spectrum are given in eV.

**Figure 3.**(

**a**) Comparison of the hybrid-Wigner-MD spectrum and its energies at the maxima (green) with the @100 QM(B3LYP)/MM-MD (blue) and with the experimental absorption spectrum of mTHPC in methanol (black). (

**b**) Comparison between QM(wB97XD)/PCM@Wigner (pink) and QM(wB97XD)/MM@hybrid-Wigner-MD (green) absorption spectra with respect to the experimental absorption spectrum of mTHPC (black) scaled with respect to the Soret band. The inset zooms the Q’ and Q′′ bands.

**Figure 4.**Comparison against the experimental reference (black) of the absorption spectra of mTHPC in methanol scaled with respect to the Soret band and computed from geometries obtained from @single-QM(B3LYP)/MM-MD simulation (orange) and from @100-QM(B3LYP)/MM-MD simulations (blue). The inset zooms the Q’ and Q’’ bands.

**Figure 5.**(

**a**) Density of states (DOS) of the first triplet excited state T

_{1}of mTHPC and the corresponding absorption spectrum obtained with the QM(wB97XD)/MM@Classical-MD (red curve for the absorption spectrum and wine for the DOS) and the QM(wB97XD)/MM@100-QM(B3LYP)/MM-MD approaches (blue curve for the absorption spectrum and cyan curve for the DOS). The centre of the T

_{1}DOS is also reported in eV. (

**b**) Absorption spectrum obtained with the QM/MM-MD sampling using B3LYP (blue) and wB97XD (grey) in the QM part. The DOS for the first triplet excited state calculated with the QM(wB97XD)/MM@100-QM(wB97XD)/MM-MD and the value at its centre is reported in purple.

**Table 1.**Excitation energies in eV of the lower lying singlet and triplet states of mTHPC computed with the static approach at the QM(wB97XD)//QM(wB97XD) and the QM(wB97XD)//QM(B3LYP) level of theory. The energy differences between the two methods is also reported in eV.

QM(wB97XD)//QM(wB97XD) | QM(wB97XD)//QM(B3LYP) | ΔE (eV) | |
---|---|---|---|

S_{1} | 2.21 | 2.01 | 0.20 |

S_{2} | 2.60 | 2.39 | 0.21 |

S_{3} | 3.40 | 3.28 | 0.12 |

S_{4} | 3.50 | 3.36 | 0.14 |

S_{5} | 4.14 | 4.10 | 0.04 |

S_{6} | 4.36 | 4.27 | 0.09 |

T_{1} | 1.07 | 0.92 | 0.15 |

T_{2} | 1.61 | 1.16 | 0.45 |

T_{3} | 2.05 | 1.98 | 0.07 |

T_{4} | 2.44 | 2.33 | 0.11 |

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**MDPI and ACS Style**

De Vetta, M.; Baig, O.; Steen, D.; Nogueira, J.J.; González, L.
Assessing Configurational Sampling in the Quantum Mechanics/Molecular Mechanics Calculation of Temoporfin Absorption Spectrum and Triplet Density of States. *Molecules* **2018**, *23*, 2932.
https://doi.org/10.3390/molecules23112932

**AMA Style**

De Vetta M, Baig O, Steen D, Nogueira JJ, González L.
Assessing Configurational Sampling in the Quantum Mechanics/Molecular Mechanics Calculation of Temoporfin Absorption Spectrum and Triplet Density of States. *Molecules*. 2018; 23(11):2932.
https://doi.org/10.3390/molecules23112932

**Chicago/Turabian Style**

De Vetta, Martina, Omar Baig, Dorika Steen, Juan J. Nogueira, and Leticia González.
2018. "Assessing Configurational Sampling in the Quantum Mechanics/Molecular Mechanics Calculation of Temoporfin Absorption Spectrum and Triplet Density of States" *Molecules* 23, no. 11: 2932.
https://doi.org/10.3390/molecules23112932