Strategy for Engineering High Photolysis Efficiency of Photocleavable Protecting Groups through Cation Stabilization

Photolabile protecting groups (PPGs) enable the precise activation of molecular function with light in many research areas, such as photopharmacology, where remote spatiotemporal control over the release of a molecule is needed. The design and application of PPGs in recent years have particularly focused on the development of molecules with high molar absorptivity at long irradiation wavelengths. However, a crucial parameter, which is pivotal to the efficiency of uncaging and which has until now proven highly challenging to improve, is the photolysis quantum yield (QY). Here, we describe a novel and general approach to greatly increase the photolysis QY of heterolytic PPGs through stabilization of an intermediate chromophore cation. When applied to coumarin PPGs, our strategy resulted in systems possessing an up to a 35-fold increase in QY and a convenient fluorescent readout during their uncaging, all while requiring the same number of synthetic steps for their preparation as the usual coumarin systems. We demonstrate that the same QY engineering strategy applies to different photolysis payloads and even different classes of PPGs. Furthermore, analysis of the DFT-calculated energy barriers in the first singlet excited state reveals valuable insights into the important factors that determine photolysis efficiency. The strategy reported herein will enable the development of efficient PPGs tailored for many applications.


Introduction
Photolabile protecting groups (PPGs) are chemical moieties that can be synthetically introduced to a substrate of interest, and subsequently be removed simply by irradiation of light of a suitable wavelength. Due to this unique property, they are widely used to remotely control the concentration of molecules with a particular function of interest (payloads), [1][2][3][4][5][6] because they allow for high spatiotemporal control over the payload release. PPGs have proven their utility in a variety of fields, ranging from molecular biology, organic synthesis to materials science. [7][8][9][10][11][12] A critical property fundamental to the applicability of PPGs is the efficiency of payload release. This efficiency is captured in the mathematical product of two factors: i) the ability of the PPG to absorb photons at the irradiation wavelength (represented by the molar attenuation coefficient ε, M -1 cm -1 ); and ii) the photochemical uncaging process quantum yield (φ, QY). In recent years, significant progress has been made into improving the first parameter, especially directed at the development of PPGs with high ε at bathochromically shifted irradiation wavelengths. [13][14][15][16][17][18][19] However, thus far enhancing the second parameter -the photochemical QY -has remained challenging. Given the importance of QY in the overall efficiency of uncaging, a general strategy for reliably increasing QY is needed, and this would represent a major step forward for the field of PPG development.
The importance of photochemical quantum yield is exemplified in the field of photopharmacology, where PPGs are used to suppress the activity of a bioactive payload in the covalently bound photocaged form. Subsequent on-site release of the bioactive molecule is achieved through lightirradiation, preventing systemic side-effects in non-irradiated areas. [1,2,[21][22][23][24][25] The importance of molar absorptivity (ε) has been widely recognized in photopharmacology and has led to impressive progress for red-or green-light responsive PPGs featuring high ε, such as those derived from the BODIPY and Coumarin scaffolds. [14,[17][18][19]26,27] However, in most cases, high ε at long irradiation wavelengths was achieved at the expense of photochemical QY, negatively affecting the overall efficiency of these PPGs. [20] Few exceptions to this trend often rely on the introduction of extended π-systems with strongly electron-donating moieties into the PPG structure, yet these strategies come at a great synthetic step cost and the flat, extended π-systems suffer from low aqueous solubility, restricting their use in photopharmacology. [28,29] Here, we present a rational strategy for increasing PPG efficiency, hinging on detailed investigation into the important factors that determine PPG QYs. Taking fundamental organic chemistry principles as the starting point, and supporting them with a DFT computational approach, we focused on 7diethylaminocoumarins as a model class of PPGs. The synthetic accessibility of coumarins and their visible light responsiveness, [12,30] has led to their privileged use for the photocaging of various functionalities, such as carboxylic acids, alcohols, and amines (as their carbonate and carbamate esters respectively), [31] making them an ideal showcase for our strategy. Furthermore, we also show that our strategy is general, and extends to other classes of PPGs that utilize heterolytic bond cleavage in their photochemical step. Finally, we demonstrate how the fluorescence increase that we observe during the uncaging of the new subtype of PPGs introduced here can be used as a convenient readout of the progress of the uncaging process in a complex biological system.

Mechanism of coumarin PPG photocleavage
The mechanism of photocleavage of Coumarin PPGs relies on heterolytic bond cleavage in the first singlet excited state (S1). [31] After photon absorption to S1, weakening of the bond between the payload and the α-carbon allows for the excited state breaking of this bond (scheme 1, k1). [31,32] Upon breakage of the payload-PPG bond, a contact ion pair (CIP) intermediate is formed, in which the positive charge is located on the coumarin chromophore and the negative charge on the payload. [31][32][33][34] The formed CIP intermediate can now undergo two different processes: i) the unproductive process, i.e., recombination to reform the substrate (scheme 1, red k-1); or ii) the productive process that results in payload release: diffusion to a Solvent Separated Ion Pair (SSIP, not shown) accompanied by water trapping to quench the carbocation (scheme 1, both processes combined in blue k2). Lowering the energy of the CIP through the use of payloads that effectively stabilize the generated negative charge (i.e. payloads with a low pKa of their conjugate acid) has been shown to improve photochemical QY. [32] However, engineering an increase of the QY in this manner is impractical, or often even impossible, since the stability of the negative charge is inherent to the payload and cannot be conveniently engineered without compromising other payload chemical properties (e.g. its bioactivity). On the other hand, the stabilization of the other component of the CIP, the PPG chromophore cation, represents an underexplored and potentially superior approach, in that it would offer a general strategy for increasing the QY of PPGs that rely on heterolytic bond cleavage, independent of the chemical nature of the payload.

Design
Although in coumarin PPGs CIP formation is a fast process, recombination of the CIP (k-1) is significantly faster than the competing productive pathway k2. [32] Based on this observation, it was deduced that rather than promoting heterolysis towards CIP formation, prevention of CIP recombination itself would be a novel strategy to significantly increase PPG photochemical QY. In the coumarin photocleavage process, the positive charge on the α-carbon (adjacent to the payload, scheme 1) in the CIP intermediate cannot be delocalized throughout the coumarin ring, as is thus fixed at the primary benzylic methylene. Being a primary cation, it is expected to be high in energy, favoring CIP recombination back to the substrate rather than the productive step (figure 1a, k-1 and k2 respectively). Based on this simple analysis, we sought to increase photolysis efficiency through direct stabilization of this cationic intermediate, thereby retarding its unproductive recombination by increasing the kinetic barrier for this process (figure 1a vs 1b, k-1). We hypothesized that the rate of CIP diffusion followed by water trapping (figure 1, k2) depends on constant coulombic forces, and will be independent of overall CIP stability. Therefore, while stabilization of the cationic intermediate will increase the barrier of the unproductive step k-1, it will not increase the barrier of the productive step k2. Consequentially, it will result in a higher ratio of the rates of k2 over k-1, increasing the overall PPG photochemical QY (figure 1b).
In fact, intermediate cation stabilization through hyperconjugation via methyl substitution at the carbon bearing the payload to produce 'secondary coumarins' has been previously attempted, but only showed marginal improvements in quantum yield. [35] Consequently, given the stability of allylic cations, we hypothesized that installing an allylic system onto the coumarin α-carbon (Figure 1c, Allylic) should significantly stabilize the incipient positive charge formed upon photoheterolysis. Since the double bond in the allylic substituent is not in conjugation with the chromophore, its influence on photochemical and photophysical properties would likely be restricted to CIP cation stability after photoheterolysis, while the wavelength of absorption and the molar attenuation coefficient should be relatively unaffected. Methyl mono-or di-substitution on the allylic system (figure 1c, 2 and 3) was expected to confer extra stabilization through hyperconjugation and further increase cation stability.
In a similar fashion, we hypothesized that direct geminal dimethylation of the α-carbon itself would stabilize the incipient CIP tertiary cation through hyperconjugation (figure 1c, Tertiary, 4), in line with results we reported earlier this year. [19]

DFT calculations
Density Functional Theory (DFT) calculations were performed to study the effect of the nature of the allylic or tertiary cations on the calculated energy barriers of CIP formation and recombination in the first singlet exited state (S1, figure 2, ΔGk1 and ΔGk-1 respectively). In all computed structures, acetic acid was chosen as the simple model payload. Geometries were optimized at the MN15/Def2SVP/SMD=H2O level of theory, [36][37][38] and the energy barriers shown are extrapolated from the ZPE-corrected free energies obtained at the same level of theory. For the designed compounds 1-4 (figure 1c), the barrier of CIP recombination was calculated to be significantly increased as compared to a reference primary coumarin with the same acetic acid payload (figure 2, red bars). The extent of increase followed an expected trend depending on the degree of hyperconjugation and delocalization.
Cation stabilization through allyl substitution on coumarins 1-3 was found to increase the energy barrier of CIP recombination more than hyperconjugation did in tertiary coumarin 4. Although the heterolysis barrier k1 also differed depending on the substituents on the α-carbon (figure 2, grey bars), less variation was observed in the energy levels of this barrier. The k1 barriers for allylic coumarins 1-3 were predicted to be energetically nearly identical (figure 2, grey bars).

Synthesis
Encouraged by the DFT calculations that predicted significantly increased energy barriers for CIP recombination in the designed coumarin-based PPGs, we set out to synthesize compounds 1-4 and study their photochemical properties. The newly designed photocages were all synthesized through a previously known aldehyde intermediate 5. [39] Reactions of this aldehyde with a variety of allylic Grignard-reagents gave the three allylic coumarin alcohols with varying methyl substitution at the northern sp 2 -carbon (scheme 2). The model photocage -primary coumarin-alcohol-is generally also obtained through one synthetic step from the same aldehyde, highlighting the synthetic accessibility of allylic coumarins 6-8. Subsequently, the allylic coumarin alcohols were loaded with the model payload acetic acid through acetylation with acetic anhydride in the presence of DMAP as a nucleophilic catalyst. The synthesis of tertiary coumarin 4 started from secondary coumarin alcohol 9, which was oxidized towards the ketone and treated with a methyl Grignard-reagent to produce the tertiary alcohol 10 (scheme 2). Tertiary alcohol 10 was loaded with the same model payload acetic acid through acetylation to yield coumarin 4.

Photochemistry
With the synthesized compounds 1-4 in hand, we sought to explore their photochemical properties using UV-Vis spectroscopy and 1 H-NMR analysis. Gratifyingly, irradiation (λ = 400 nm) of 1-4 followed by 1 H-NMR analysis confirmed that all compounds released the payload acetic acid upon irradiation  To rationalize these results, the observed heterolysis QYs were compared to the heterolysis energy barriers of CIP formation and recombination as calculated by DFT. Gratifyingly, we found that the barrier height of the unproductive k-1 process (the CIP recombination) was a good predictor for the quantum yield (SI, Figure S86). In contrast, we observed that the calculated energy barrier for the k1 process correlated poorly to photochemical QY (figure 3f). These findings confirm our initial hypothesis that the limiting factor in coumarin photochemical QY is CIP recombination, rather than CIP formation itself. Also, the relative barrier height ratio of k-1 over k1 showed a strong correlation with photochemical QY, exhibiting an exponential relationship between this relative barrier height ratio and the QY (figure 3g). These results demonstrate that retarding CIP recombination has resulted in drastic enhancements in photochemical QY, and moreover confirmed this as a viable strategy for improving PPG efficiency.
A possible explanation for the poor correlation of k1 barrier height to the QY could be that this barrier is partially overcome through excitation to a higher vibrational level in S1, regardless of the nature of the incipient CIP cation. This hypothesis is supported by the large Stokes-shift of 7-diethylaminocoumarins (SI, figure S49), suggesting a higher S1 vibrational level is almost always accessed upon excitation. Although the seemingly important productive forward step k2 barrier height is not included in this calculation, our calculations nonetheless provide an exponential trend correlating reversibility of CIP formation to QY. This suggests that the k2 barrier height is energetically comparable for all shown coumarin photocages, also confirming our initial hypothesis that this barrier is largely independent of CIP stability. This is plausible, since both the acetate payload and the photoproduct cations in the CIPs formed from the photo-heterolysis of each PPG variant 1-4 are similar in their electrostatic topology, and thus must be similar in their Coulombic nature. Thus, the diffusion rate out of the CIP, ultimately resulting in productive cation trapping by the solvent, would be expected to be near-identical for each PPG variant 1-4.
The photochemical quantum yield of tertiary coumarin 4 (figure 3g, dark blue) did not fit the predicted trend. This discrepancy can be explained by the fact that tertiary coumarin 4 exhibits a different mechanism of CIP escape. Whereas the usual mechanism of CIP escape is diffusion followed by water trapping, tertiary coumarin exhibited elimination towards an alkene (figure 3b). Since the photochemical quantum yield of tertiary coumarin is higher than expected (i.e. can be found above the curve fit) it can be concluded that its deprotonation barrier must be lower than the k2 barrier for the Allylic-coumarins 1-3 and primary-and secondary-coumarins. Currently we are performing further studies on the effect of the k2 barrier on PPG photoheterolysis efficiency.
In literature describing the coumarin PPG mechanism of photocleavage, often CIP recombination and relaxation to S0 is reported as a concerted process (i.e. a conical intersection of the S1 and S0 potential energy surfaces exists at the CIP recombination event). [31,32] However, one must also consider the possibility that relaxation back to S0 happens before CIP recombination, and thus that CIP recombination happens on the S0 potential energy surface. We could not find definitive evidence for this situation, but nonetheless we attempted to calculate the energy barriers for CIP recombination on the S0 potential energy surface as well. Our DFT calculations we were unable to find a stable CIP in the S0 state, or even a heterolysis transition state for all described coumarins 1-4. Although we can't fully exclude some extent of recombination occurring in the ground state, the tight correlation of the measured QYs with the S1 energy barriers suggests that CIP recombination in S1 seems the most reasonable.

Allylic substitution as a general strategy for quantum yield enhancement
Since the photochemical QY improvement of coumarin photocages was achieved through the stabilization of the cationic chromophore component of the CIP, we predicted that it would be a general strategy for QY improvement that would also hold for payloads other than carboxylic acids. To explore this generality, the allylic PPG with the highest QY (compound 3) was also used to cage and release another moiety that is often photocaged in bioactive payloads: an amine group. Due to their inability to stabilize a negative charge, amines are generally photocaged as their carbamate analogues. [40] Two photocages bearing an amine payload were synthesized, one bearing the allylic substituent on the alpha-carbon ( figure 4a, 12, red) and a primary reference compound ( figure 4a, 11, black). Upon comparing their photochemical quantum yields, we were pleased to find that the allylic carbamate also displayed very efficient photolysis. Its quantum yield underwent a >35 fold improvement over that of the model primary coumarin bearing the same carbamate payload, an even more pronounced effect than the one that was observed for acetate payloads. These results highlight the robustness of this approach: PPG cation stabilization is a general way to improve photochemical QY for a diverse subset of payloads.
Finally, we aimed to see if this optimization strategy holds also for a different member of the class of heterolytic PPGs. Arguably, all PPGs belonging to this class would benefit from a stabilized incipient chromophore cation through allylic substitution at the α-carbon. To illustrate this, we set out to introduce allylic substitution on another widely used class of photocages, BODIPY PPGs. Allylic-BODIPY PPG 14 (figure 4b, blue) was synthesized and confirmed to release acetic acid upon irradiation (SI, section 7). Interestingly, rather than photobleaching that is often observed for BODIPY photocages, Allylic-BODIPY PPG 14 showed clean conversion to a different BODIPY-photoproduct with a clear isosbestic point (figure 4b). The photoproduct is likely the result of water trapping of the cation ( 1 H-NMR, SI figure S60). Finally, the rate of deprotection of Allylic-BODIPY PPG 14 was compared to that of primary BODIPY 13 and a sevenfold improvement in rate was found. These findings illustrate the versatility of this simple principle: our strategy of cation stabilization results in significant improvements in photochemical QY and holds for multiple types of PPGs.

Fluorescence properties of the coumarin uncaging product
In the photoheterolysis process, the allylic coumarin  irradiation can be used to follow the payload release through normalization of the fluorescence signal. [41][42][43] To illustrate the utility of the allylic PPGs 1-3 and their fluorescence properties, we aimed to use the PPGs to photocage a model bioactive compound and release this compound upon irradiation in a process whose progress could be independently followed solely by fluorescence. Due to our lab's experience in the photoactivation of antibiotics, [44][45][46][47] we have chosen piperacillin -a potent member of the beta-lactam class -as a model bioactive compound.
Since the caged antibiotics were to be used in a 24 h bacterial growth assays, the hydrolytic stability of the model photocaged compounds 1-3 was first evaluated. Allylic photocages 1 and 2 showed excellent hydrolytic stability, forming no hydrolysis product after 15 h at 25 °C in the dark. Allylic photocage 3, however, showed slight hydrolysis after 15 h, likely by virtue of its superior cation stability after hydrolysis (SI, figure S82). Given its greater hydrolytic stability, it was decided to use allylic photocage 1 for the construction of the caged antibiotics whose activation can be followed by fluorescence. In an initial assay, the minimal inhibitory concentration (MIC) of piperacillin towards E. coli CS1562 was determined to be 0.5-1.0 µM (SI, figure S77). The required photocaged piperacillin 16 (figure 6a) was conveniently obtained through a Steglich esterification of Piperacillin with the corresponding allylicalcohol 6.
Irradiation of photocage 16 showed the expected emergence of a fluorescent response due to double bond rearrangement (figure 6b). The predicted degree of payload release was calculated from the fluorescence signal through an exponential decay curve fit and was corrected for the observed amount of payload rearrangement (10 %, UPLC-MS, SI). To our delight, the predicted concentrations of the liberated piperacillin (obtained after irradiation with  = 400 nm light for much less than a minute) corresponded perfectly to bacterial growth inhibition (Figure 6c These results confirm the practical utility of the allylic photocages reported herein and illustrate the reliability of the fluorescent signal that can be used to quantitatively predict the amount of bioactive payload released in biological assays. In sum, our newly developed PPGs 1-3, developed using the QYenhancement strategy described herein, allowed for the rapid release of complex biological payloads in complex biological environments, all while enabling a convenient fluorescence readout method for monitoring the uncaging process.

Conclusion and Outlook
In this study, we identify an approach of preventing CIP recombination in PPG photolysis as a key strategy to greatly increase the photolysis QY. The QY enhancement strategy is supported by DFT calculations which predict higher energy barriers for the recombination process. An excellent correlation between the reversibility k-1 of the heterolytic step and the photochemical quantum yield was observed. We also concluded that a poor correlation exists between the calculated k1 barrier and our measured QYs and have thus demonstrated that preventing recombination of the CIP, rather than kinetically stimulating its formation, is a crucial strategy in increasing PPG efficiency. Furthermore, we showed that our strategy for increasing PPG efficiency through incipient cation stabilization is general for different payloads (carboxylic acids and amines) and for different PPG classes (coumarin and BODIPY PPGs), again demonstrating the versatility of this simple principle.
Given their synthetic accessibility and high photochemical QYs, the efficient PPGs 1-3 themselves, designed applying the cation stabilization strategy showcased herein, could prove valuable in assays that require near-instant release of a payload, such as those in time-resolved structural biology studies. [48] Furthermore, the fluorescent readout of PPGs 1-3 can be reliably used to quantify the amount of complex biological payload release, in complex biological environments, as is also demonstrated.
We believe the new PPG design strategy demonstrated herein directly addresses the need for increasing PPG efficiency in the rapidly expanding research fields that rely on precise activation of molecular function with light. Our QY engineering strategy will undoubtedly prove valuable for the development of ideal photocages tailored for more complex applications, for example (but not limited to) red-shifted photocages with high quantum yields for use in photopharmacology.