Competition Between Cyclization and Unusual Norrish Type I and Type II Nitro-Acyl Migration Pathways in the Photouncaging of 1-Acyl-7nitroindoline Revealed by Computations
The 7-nitroindolinyl family of caging chromophores has received much attention in the past two decades. However, its uncaging mechanism is still not clearly understood.In this study, we performed state-of-the-art density functional theory calculations to unravel the photo-uncaging mechanism in its entirety, and we compared the probabilities of all plausible pathways. We found competition between a classical cyclization and acyl migration pathways, and here we explain the electronic and steric reasons behind such competition. The migration mechanism possesses the characteristics of a combined Norrish Type I and a 1,6-nitro-acyl variation of a Norrish Type II mechanism, which is reported here for the first time. We found out that the improved quantum yields of the 4-methoxy-5,7-dinitroindolinyl (MDNI) cages, compared
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<p>to their mononitro analogues (MNI), is due to faster intersystem crossing. Our findings may pave the way for improved cage designs that possess higher quantum yields and a more efficient agonist release.</p><p>The ability to deliver or activate a compound to a specific site with precise timing is of extreme value. This is widely referred to as spatio-temporal control. [1][2][3][4] Techniques that possess spatio-temporal advantages have found numerous applications ranging from medicinal chemistry fields with photo targeted therapeutics, 1,3 such as cancer treatments [5][6][7][8] or photoinitiated catalysis, 8 and to the neuroscience where it becomes critical to activate selected neurons to study specific pathways. 1,[9][10][11][12] In the latter, emerging field of optogenetics, light-sensitive channel rhodopsin receptors are genetically engineered in selected neurons rendering them light responsive. 3,11,[13][14][15][16][17][18][19] Additional methods of firing specific neurons employed a diverse array of photocleavable protecting groups attached to critically active moieties of agonists. [20][21][22][23] These photocleavable protecting groups acquired the term photocages, or simply cages, as their attachment to an agonist necessarily incapacitates it, much like caging a ferocious animal.</p><p>Caged molecules are inert, and they need an external agent-such as a physical, chemical, or mechanical force- 8,[24][25][26][27] to induce the release of the biologically active compound they are protecting. Light represents the most advantageous activator because it offers the ability to control the precise time and the location of the release. [1][2][3][4] Although there are numerous classes of photocleavable groups, they share a common feature of absorbing light of specific wavelengths that leads to excitation from the singlet ground state (S0) to an excited singlet state (S1,2,3…), followed by an intersystem crossing (ISC) to the triplet state manifold, and consequential fast relaxation to the lowest triplet state (T1). 21,28,29 This leads to cleavage of the protecting group at the most labile bond, which is critically positioned for such release (as shown in red for 4-methoxy-5,7-dinitroindolinyl glutamate (MDNI-Glu), in Figure 1 below). These mechanisms depend on the class of protecting groups employed, but some are not clearly understood, as mechanisms in excited states may not resemble the familiar mechanistic pathways commonly accepted for ground states. 21 7-Nitroindolinyl cages have gained popularity at the turn of the century due to their optimal quantum yields and efficient release of agonists. [30][31][32] Improvements in such cage systems led to the installation of a second nitro group at the 5 position that resulted in improved quantum yields. 33,34 Although the second nitro at the C-5 is not directly participating in the mechanism, as we will show later, its presence results in an improved quantum yield by at least fivefold according to experimental data. [35][36][37] The mechanism of uncaging for the 7-indolinyl system was studied by at least three groups using kinetic, fast pulse IR, and computation. 33,38,39 However, the most recently reported computational mechanisms of uncaging do not explain the difference in quantum yield, which is an important parameter, especially for consideration in future improved designs. We herein report a thorough computational analysis that highlights the electronic effects behind the better quantum yields of dinitroindolinyl systems and also clarifies the mechanism with respect to the traditionally accepted formation of a cyclic intermediate.</p><p>The first mechanism for the light-initiated uncaging of MDNI-Glu (1 in Scheme 1) has been proposed by Ellis-Davies and coworkers in 2005, 34 and is based on kinetic data collected by Morrison et al. 40 for another member of the 7-nitroindoline family. For simplicity, we will refer to this mechanism as the cyclization pathway (CP) from here on. According to their proposed mechanism, after irradiation, the reaction proceeds on the triplet surface via a cyclic intermediate (5). Subsequently, the system is deprotonated (3), and finally, it delivers the free glutamate (7) to the reaction medium. According to the CP mechanism, the increased reactivity of MDNI is due to the influence of the nitro group in position 5 on the overall electronic structure of the indoline scaffold. 34 A subsequent computational study by Pálfi et  In this work, we aim to redeem the controversy by performing state-of-the-art density functional theory (DFT) calculations of the full reaction mechanism with the ωB97X-D exchange-correlation functional approximation 41 and the def2-TZVP basis set 42 on top of geometries optimized at the B3LYP-D3(BJ)/def2-SVPD level of theory. [42][43][44][45][46] We chose the</p><!><p>ωB97X-D method based on the fact that it reproduces the experimental UV-Vis spectra of both 4-methoxy-7-nitroindolyl (MNI) and MDNI (vide infra), and we validated its results using several other exchange-correlation functionals (ωB97M-V, M11, MN15, DSD-PBEP86-D3(BJ), LC-ωHPBE and CAM-B3LYP, see section S2 of the Supporting Information for details). The B3LYP-D3(BJ)/def2-SVPD method was used for geometry optimization of all species because it was shown to produce reliable geometries for several systems in previous studies. [47][48][49] To address the potential multi-reference character of some of the species on the excited state triplet surface, we also used the B1 50 and Aλ 51 diagnostics, which showed a lack of strong multi-reference character for all the relevant transition structures (see sections S4 and S8 and Tables S1 and S2 in the Supporting Information). We calculate and also report for the first time the Gibbs free energy profile of the reaction, including both enthalpic and entropic corrections. For computational efficiency, we first focused our attention on MDNI acetate (MDNI-Ac) and MNI acetate (MNI-Ac), two systems that closely resemble the original MDNI-Glu and MNI-Glu systems reported in the literature. Our results show that there is a competition between the cyclization and migration pathways, but they do not significantly differ from each other. The extension of the study to the relevant structures of the larger MDNI-Glu and MNI-Glu systems shows very little difference in the reactivity of the glutamate with respect to the acetate.</p><p>The cyclic intermediate proposed by Ellis-Davies indeed forms, and it represents the lowest-energy pathway in the overall reaction. However, the difference with the migration mechanism is negligible, ~2.6 kcal/mol calculated at the ωB97X-D/def2-TZVP level of theory.</p><p>Additional calculations on the MDNI-Glu and 𝛼-MDNI-Glu systems suggest that the different substituents do not affect the mechanism of the reaction on the triplet surface contrarily to what has been previously reported. 39 The cyclization and migration steps are within 2.3 kcal/mol from each other independently of the way glutamate is bound to the cage scaffold. This amount is very close to the one we obtained for MDNI-Ac (vide infra), so we concluded that the uncaging mechanism is not affected by the length of the side chain, nor by its nature. Also, the second nitro group in position 5 does not affect the pathway significantly. The differences we found (Table S3 in the Supporting Information) are within ~4.0 kcal/mol of each other, and the highest activation energy for the migration pathway is 10.8 kcal/mol, while it is 8.2 kcal/mol for the cyclization pathway. Such barriers do not represent an obstacle to the reactivity of the molecule, as they are easily overcome at room temperature. Having excluded any significant effect from the substituents to the sensitivity of the molecule towards light absorption, other reasons lie behind the differences in quantum yields. We contend that the second nitro group enables a faster population of the triplet state, thus increasing the overall quantum yield of the MDNI-based cages over their mononitro analogues. We investigated this idea through the estimation of the rate constants of intersystem crossing (which has never been done previously for this class of compounds). Validation of the results using the available experimental data confirms our hypothesis, and it shows promise for future application to the prediction of quantum yields of molecules that have not been previously synthesized.</p><p>The reaction from MDNI-Ac (10 in Scheme 2) to 4-methoxy-5-nitro-7-nitrosoindole (8)   and acetic acid is thermodynamically favored, with a calculated ΔG of -35.8 kcal/mol (see Scheme 2 below). According to the CP mechanism, the cyclic intermediate 9 (see below) is the key structure in the reaction mechanisms of MNI and MDNI, regardless of the leaving group.</p><p>Close inspection of its structure reveals that there are at least four bonds (labeled A, B, C, or D in structure 9) that can potentially break, leading to four different routes depending on which of them breaks first. We will label these routes according to the bond that breaks first. The migration pathway necessitates only one step (structure 11 in Scheme 2) to yield the nitronic anhydride 13, while the cyclization pathway requires two consecutive steps-the formation and the subsequent opening of the cyclic intermediate 9. The overall energy required to go through these steps is similar, albeit slightly smaller for the cyclization, as seen in Scheme 2. In order to rationalize this discovery, we looked at a few structural features of the corresponding transition structures (TSs, structures 11 and 12), as reported in Table 1.</p><p>Specifically, we considered the bond lengths of bond A and bond C (see Figure 1), in conjunction with the bond angle they describe. On the one hand, the bond angle is very similar for the two TSs, differing by only 3.7 degrees. This fact shows that the molecule necessitates deplanarization before undergoing any additional change, which exploits the favorable interaction between the available unoccupied orbital of the carbonyl and the singly occupied orbital of the oxygen radical regardless of the pathway (Figure 2). If the carbonyl group is substituted with an ethyl group, the cyclization pathway is no longer accessible. The migration becomes the only possible pathway, although the electronic effects that make it more accessible are lost (vide infra).</p><p>On the other hand, the two bond lengths differ slightly in the two transition structures.</p><p>They are shorter for the cyclization TS, which justifies why the cyclization process is slightly more favored than the migration process. The acetyl group needs to move farther away from the cage when the migration process occurs, while in the cyclization pathway, the oxygen radical can readily attack the carbon atom without undergoing substantial modifications. The energy requirements for the two reactions are consistent with this concept. The migration process has a slightly larger barrier (5.8 kcal/mol) because the transition structure requires a more significant rearrangement than the cyclization process, which has shorter bonds and a less strict geometric requirement. The cyclization requires 4.7 kcal/mol overall (3.3 kcal/mol for the first step, and an additional 1.4 kcal/mol for the ring-opening).   52,56 In order to better understand this migration pathway, we computed the energy for an ethyl group migration (in place of the acetyl) and noted that it was higher in energy (21.3 kcal/mol, Figure 3 part c), despite being positioned in a 1,6-relationship to the nitro oxygen radical. This reflects the stabilizing effect of an acyl radical in the transition state of the 1,6-migration. The energy difference between the migration and the cyclization pathways is calculated at 1.1 kcal/mol, and since the expected accuracy of our calculations is ±1.0 kcal/mol, [57][58][59][60][61] we can conclude that the two mechanisms are competitive. As such, given the small activation barriers, both mechanisms can co-occur. We found the same to be true for MNI-Ac as well, with the cyclization requiring 4.8 kcal/mol overall, and the migration needing 5.7 kcal/mol.  Furthermore, the hydrolysis product (15) and the uncaging product (8) are different in that the former retains the nitro group while the photo uncaging leads to the nitroso. However, both processes result in the release of acetic acid (from 10 and 13, respectively).</p><p>In summary, our improved computational protocol on the uncaging mechanism show that the lowest energy path proceeds as follows. After irradiation, the first step is either the migration of the acetyl group from the nitrogen atom of indoline to one of the oxygen atoms of the neighboring nitro group (blue line in Scheme 3), or the formation of the cyclic intermediate and its subsequent opening (orange line in Scheme 3). The migration transition structure (structure 18) is only 5.8 kcal/mol higher than the reactant, and it is accessible at room temperature. The cyclization step involves two subsequent reactions having an activation barrier of 3.3 and 1.4 kcal/mol, respectively (structures 19 and 21 in Scheme 3). In all cases, the energy barriers are non-prohibitive, making both routes plausible at room temperature. Both pathways converge towards the formation of the nitronic anhydride (structure 22). The subsequent step ( 23) has a barrier of 8.1 kcal/mol. After the leaving group (acetate in this case) is released (23), a charged nitroso intermediate then forms (24), which readily deprotonates and then dis-excites, going back to the singlet surface as a spent cage (25). This is also consistent with the fact that molecules including an aromatic ring are more acidic in the excited state than in the ground state. [62][63][64][65] Subsequently, the non-aromatic five-membered ring in 25 spontaneously tautomerizes to the fully aromatic indole system of 4-methoxy-5-nitro-7-nitrosoindole (26). The reaction mechanism for MDNI-Ac is outlined in Scheme 3. Having excluded the uncaging mechanism on the triple surface as a potential explanation of the quantum yield, the most probable steps that can affect the behavior of this family of molecules are the response towards the incident light and how readily the triplet state is  A singlet-triplet transition is strictly forbidden in non-relativistic quantum mechanics, but it can happen, in practice, because of intersystem crossing (ISC). Higher quantum yields generally correspond to higher rates of ISC, as established, for example, for the borondipyrromethene (BODIPY) family of cages. 23,67 We expect a similar trend for the MNI and MDNI cages, but a characterization of the process for these molecules has not been reported to date. We report a qualitative description of the process, with preliminary quantitative results that match the experimental observations. This procedure can also be extended-upon further validation-to the prediction of relative quantum yields of newly synthesized cages, and it can provide valuable information for the design of new molecules.</p><p>Borrowing from the work of Russo, Chiodo, Thiel, and coworkers, 68,69 we estimated the rate constant for the ISC process for each state, 𝑘 !"# $% as:</p><p>where λM is the Marcus reorganization energy, ∆EST the energy difference between the n-th singlet excited state and the m-th triplet state, SOCnm the magnitude of spin-orbit coupling (SOC) between states n and m. 70 (T=298.15 K; R, and h are the ideal gas and Planck's constants, respectively.) We calculated the excitation energies, ∆EST, of the first ten excited singlet states and the first ten excited triplet states with TD-DFT using the ωB97X-D functional and the def2-TZVP basis set. The SOC matrix elements involving the coupling of all the singlet states with all the triplet states were also calculated using the one-electron Breit-Pauli Hamiltonian, as implemented in the Q-Chem code.</p><p>Both MDNI and MNI have three singlet-singlet transitions in the relevant region of the UV-Vis spectrum (250 -500 nm, Figure 4). However, our results show that the transitions with the largest ISC involve the first singlet state exclusively. Calculations of the Marcus reorganization energies for each excited state are non-trivial-especially when the second and higher states are involved-and are beyond our current capabilities. However, typical observed values of λM are in the range between 0.1 eV and 0.2 eV. 71,72 The overall KISC is calculated as the sum of all the individual 𝑘 !"# -% for all the states, while each individual 𝑘 !"# -% can be plotted as a function of ∆EST for fixed values of λM, sampled in the typical range, as in Figure 5. These plots reveal that the Marcus reorganization energies do not have a substantial influence on the overall rate of ISC for each of the individual molecules. As such, they have minimal effects on the relative trends, especially on the ratio between the two molecules' values.</p><p>The two molecules have either five (MDNI-Glu), or three (MNI-Glu) triplet states lying below the first singlet excited state. Four of these states (T2, T3, T4, and T5) give substantial contributions to the overall ISC rate constant for MDNI, while only one state (T3) contributes for MNI, regardless of the value of λM. The orbital analysis of the transitions with the largest contributions (S1 → T5 for MDNI, and S1 → T3 for MNI) reveals that they both involve a similar change in orbital type, going from a 1 (π → π*) state to a 3 (σ → π*) state for both molecules, in perfect agreement with El-Sayed's rule. 73 These results are summarized in Figure 5, where each contributing state is labelled on the λM = 0.15 curve. For this specific value of λM, we also obtain quantitative agreement with the available experimental data, 21,34,36 with an estimated value of 3.88 for the MDNI/MNI ratio, against experimental values of ~ 4 (see Section S12 of the Supporting Information). The same considerations and results apply to α-MDNI-Glu and α-MNI-Glu (Table S6), for which the calculated ratio is 4.14.</p><p>The plots in Figure 5 can also be used as a tool to design cages with better quantum yields. The key for this task is to consider modifications to the M(D)NI cage with an increased number of excited triplet states with energies lower than the first singlet excited state. In conclusion, we present a detailed description of the reaction mechanism of 4-methoxy-5,7-dinitroindolinyl acetate (MDNI-Ac) and 4-methoxy-7-nitroindolinyl acetate (MNI-Ac). We show that the mechanism proceeds in the triplet state through two competitive pathways via the formation of a cyclic intermediate or through a Norrish type I and II 1,6-acyl migration to the nitro group. We analyze the geometric and electronic features of the competitive routes. Both pathways yield a nitronic anhydride, which subsequently releases the leaving group of the nitronic ester, while the spent cage is recovered in the singlet ground state. The reason behind the superior photoproperties of MDNI as a better caging group does not lie on lower activation energies, but rather on a better sensitivity towards light due to an increased rate of intersystem crossing. This is possible because the additional nitro group on the MDNI cages lowers the energy of the triplet states with respect to the first singlet state, thus increasing their availability to undergo ISC. Our findings provide additional insights about the reactivity of the MDNI family of photocages, and guidance for the future synthesis of improved ones.</p><!><p>Computational methods and models.</p><p>Details about the computational methods, the theoretical frameworks, and the computational programs used can be found in Section S12 in the Supporting Information. The appropriate references are detailed in the Reference section (Section S15) of the Supporting Information. Last, sample input files for all calculations are also provided (Section S14 in the Supporting Information).</p>